CD40L deficiency attenuates diet-induced adipose tissue inflammation by impairing immune cell accumulation and production of pathogenic IgG-antibodies

Background

Adipose tissue inflammation fuels the metabolic syndrome. We recently reported that CD40L – an established marker and mediator of cardiovascular disease – induces inflammatory cytokine production in adipose cells in vitro. Here, we tested the hypothesis that CD40L deficiency modulates adipose tissue inflammation in vivo.

Methodology/Principal Findings

WT or CD40L^−/− mice consumed a high fat diet (HFD) for 20 weeks. Inflammatory cell recruitment was impaired in mice lacking CD40L as shown by a decrease of adipose tissue macrophages, B-cells, and an increase in protective T-regulatory cells. Mechanistically, CD40L-deficient mice expressed significantly lower levels of the pro-inflammatory chemokine MCP-1 both, locally in adipose tissue and systemically in plasma. Moreover, levels of pro-inflammatory IgG-antibodies against oxidized lipids were reduced in CD40L^−/− mice. Also, circulating low-density lipoproteins and insulin levels were lower in CD40L^−/− mice. However, CD40L^−/− mice consuming HFD were not protected from the onset of diet-induced obesity (DIO), insulin resistance, and hepatic steatosis, suggesting that CD40L selectively limits the inflammatory features of diet-induced obesity rather than its metabolic phenotype. Interestingly, CD40L^−/− mice consuming a low fat diet (LFD) showed both, a favorable inflammatory and metabolic phenotype characterized by diminished weight gain, improved insulin tolerance, and attenuated plasma adipokine levels.

Conclusion

We present the novel finding that CD40L deficiency limits adipose tissue inflammation in vivo. These findings identify CD40L as a potential mediator at the interface of cardiovascular and metabolic disease.     

Amplified B lymphocyte CD40 signaling drives regulatory B10 cell expansion in mice

Background

Aberrant CD40 ligand (CD154) expression occurs on both T cells and B cells in human lupus patients, which is suggested to enhance B cell CD40 signaling and play a role in disease pathogenesis. Transgenic mice expressing CD154 by their B cells (CD154^TG) have an expanded spleen B cell pool and produce autoantibodies (autoAbs). CD22 deficient (CD22^−/−) mice also produce autoAbs, and importantly, their B cells are hyper-proliferative following CD40 stimulation ex vivo. Combining these 2 genetic alterations in CD154^TGCD22^−/− mice was thereby predicted to intensify CD40 signaling and autoimmune disease due to autoreactive B cell expansion and/or activation.

Methodology/Principal Findings

CD154^TGCD22^−/− mice were assessed for their humoral immune responses and for changes in their endogenous lymphocyte subsets. Remarkably, CD154^TGCD22^−/− mice were not autoimmune, but instead generated minimal IgG responses against both self and foreign antigens. This paucity in IgG isotype switching occurred despite an expanded spleen B cell pool, higher serum IgM levels, and augmented ex vivo B cell proliferation. Impaired IgG responses in CD154^TGCD22^−/− mice were explained by a 16-fold expansion of functional, mature IL-10-competent regulatory spleen B cells (B10 cells: 26.7×10⁶±6 in CD154^TGCD22^−/− mice; 1.7×10⁶±0.4 in wild type mice, p<0.01), and an 11-fold expansion of B10 cells combined with their ex vivo-matured progenitors (B10+B10pro cells: 66×10⁶±3 in CD154^TGCD22^−/− mice; 6.1×10⁶±2 in wild type mice, p<0.01) that represented 39% of all spleen B cells.

Conclusions/Significance

These results demonstrate for the first time that the IL-10-producing B10 B cell subset has the capacity to suppress IgG humoral immune responses against both foreign and self antigens. Thereby, therapeutic agents that drive regulatory B10 cell expansion in vivo may inhibit pathogenic IgG autoAb production in humans.     

Mice Lacking Natural Killer T Cells Are More Susceptible to Metabolic Alterations following High Fat Diet Feeding

Current estimates suggest that over one-third of the adult population has metabolic syndrome and three-fourths of the obese population has non-alcoholic fatty liver disease (NAFLD). Inflammation in metabolic tissues has emerged as a universal feature of obesity and its co-morbidities, including NAFLD. Natural Killer T (NKT) cells are a subset of innate immune cells that abundantly reside within the liver and are readily activated by lipid antigens. There is general consensus that NKT cells are pivotal regulators of inflammation; however, disagreement exists as to whether NKT cells exert pathogenic or suppressive functions in obesity. Here we demonstrate that CD1d^−/− mice, which lack NKT cells, were more susceptible to weight gain and fatty liver following high fat diet (HFD) feeding. Compared with their WT counterparts, CD1d^−/− mice displayed increased adiposity and greater induction of inflammatory genes in the liver suggestive of the precursors of NAFLD. Calorimetry studies revealed a significant increase in food intake and trends toward decreased metabolic rate and activity in CD1d^−/− mice compared with WT mice. Based on these findings, our results suggest that NKT cells play a regulatory role that helps to prevent diet-induced obesity and metabolic dysfunction and may play an important role in mechanisms governing cross-talk between metabolism and the immune system to regulate energy balance and liver health.     

Programmed Cell Death-1 Deficiency Exacerbates T Cell Activation and Atherogenesis despite Expansion of Regulatory T Cells in Atherosclerosis-Prone Mice

T cell activation represents a double-edged sword in atherogenesis, as it promotes both pro-inflammatory T cell activation and atheroprotective Foxp3⁺ regulatory T cell (Treg) responses. Here, we investigated the role of the co-inhibitory receptor programmed cell death-1 (PD-1) in T cell activation and CD4⁺ T cell polarization towards pro-atherogenic or atheroprotective responses in mice. Mice deficient for both low density lipoprotein receptor and PD-1 (Ldlr^−/−Pd1^−/−) displayed striking increases in systemic CD4⁺ and CD8⁺ T cell activation after 9 weeks of high fat diet feeding, associated with an expansion of both pro-atherogenic IFNγ-secreting T helper 1 cells and atheroprotective Foxp3⁺ Tregs. Importantly, PD-1 deficiency did not affect Treg suppressive function in vitro. Notably, PD-1 deficiency exacerbated atherosclerotic lesion growth and entailed a massive infiltration of T cells in atherosclerotic lesions. In addition, aggravated hypercholesterolemia was observed in Ldlr^−/−Pd1^−/− mice. In conclusion, we here demonstrate that although disruption of PD-1 signaling enhances both pro- and anti-atherogenic T cell responses in Ldlr^−/− mice, pro-inflammatory T cell activation prevails and enhances dyslipidemia, vascular inflammation and atherosclerosis.     

Global deletion of MGL in mice delays lipid absorption and alters energy homeostasis and diet-induced obesity

Monoacylglycerol lipase (MGL) is a ubiquitously expressed enzyme that catalyzes the hydrolysis of monoacylglycerols (MGs) to yield FFAs and glycerol. MGL contributes to energy homeostasis through the mobilization of fat stores and also via the degradation of the endocannabinoid 2-arachidonoyl glycerol. To further examine the role of MG metabolism in energy homeostasis, MGL^−/− mice were fed either a 10% (kilocalories) low-fat diet (LFD) or a 45% (kilocalories) high-fat diet (HFD) for 12 weeks. Profound increases of MG species in the MGL^−/− mice compared with WT control mice were found. Weight gain over the 12 weeks was blunted in both diet groups. MGL^−/− mice were leaner than WT mice at both baseline and after 12 weeks of LFD feeding. Circulating lipids were decreased in HFD-fed MGL^−/− mice, as were the levels of several plasma peptides involved in glucose homeostasis and energy balance. Interestingly, MGL^−/− mice had markedly reduced intestinal TG secretion following an oral fat challenge, suggesting delayed lipid absorption. Overall, the results indicate that global MGL deletion leads to systemic changes that produce a leaner phenotype and an improved serum metabolic profile.     

Group 1B phospholipase A₂ deficiency protects against diet-induced hyperlipidemia in mice

Excessive absorption of products of dietary fat digestion leads to type 2 diabetes and other obesity-related disorders. Mice deficient in the group 1B phospholipase A₂ (Pla2g1b), a gut digestive enzyme, are protected against diet-induced obesity and type 2 diabetes without displaying dietary lipid malabsorption. This study tested the hypothesis that inhibition of Pla2g1b protects against diet-induced hyperlipidemia. Results showed that the Pla2g1b^−/− mice had decreased plasma triglyceride and cholesterol levels compared with Pla2g1b^+/+ mice subsequent to feeding a high-fat, high-carbohydrate (hypercaloric) diet. These differences were evident before differences in body weight gains were observed. Injection of Poloxamer 407 to inhibit lipolysis revealed decreased VLDL production in Pla2g1b^−/− mice. Supplementation with lysophosphatidylcholine, the product of Pla2g1b hydrolysis, restored VLDL production rates in Pla2g1b^−/− mice and further elevated VLDL production in Pla2g1b^+/+ mice. The Pla2g1b^−/− mice also displayed decreased postprandial lipidemia compared with Pla2g1b^+/+ mice. These results show that, in addition to dietary fatty acids, gut-derived lysophospholipids derived from Pla2g1b hydrolysis of dietary and biliary phospholipids also promote hepatic VLDL production. Thus, the inhibition of lysophospholipid absorption via Pla2g1b inactivation may prove beneficial against diet-induced hyperlipidemia in addition to the protection against obesity and diabetes.     

A functional role for the p62–ERK1 axis in the control of energy homeostasis and adipogenesis

In vivo genetic inactivation of the signalling adapter p62 leads to mature-onset obesity and insulin resistance, which correlate with reduced energy expenditure (EE) and increased adipogenesis, without alterations in feeding or locomotor functions. Enhanced extracellular signal-regulated kinase (ERK) activity in adipose tissue from p62-knockout (p62^−/−) mice, and differentiating fibroblasts, suggested an important role for this kinase in the metabolic alterations of p62^−/− mice. Here, we show that genetic inactivation of ERK1 in p62^−/− mice reverses their increased adiposity and adipogenesis, lower EE and insulin resistance. These results establish genetically that p62 is a crucial regulator of ERK1 in metabolism.     

T cells from Programmed Death-1 deficient mice respond poorly to Mycobacterium tuberculosis infection

Background

Programmed Death-1 (PD-1; CD279) receptor molecule is widely believed to be a negative regulator predominantly expressed by exhausted/activated mouse T cells. Upon interaction with its ligands, PD-L1 and PD-L2, PD-1 inhibits activation of T cells and cytokine production, which has been documented in various viral and fungal infections as well as in vitro studies. Therefore, inhibition of T cell responses by PD-1 resulted in disease resistance in a variety of mouse infection models studied heretofore.

Methodology/Principal Findings

Here, we report that PD-1 deficient (PD-1^−/−) mice infected with Mycobacterium tuberculosis (M. tb) H37Rv by the aerosol route have increased susceptibility as compared with their wild type littermates. Surprisingly, M. tb antigen-specific T cell proliferation was dramatically reduced in PD-1 deficient animals compared with wild-type littermates, and this was due to increased numbers of regulatory T cells (Tregs) and recruitment of mesenchymal stem cells. Furthermore, PD-1^−/− mice exhibited decreases in the autophagy-induced LC3-B marker protein in macrophages.

Conclusions/Significance

Our findings suggest that PD-1 does not play an inhibitory role during M. tb infection and instead promotes mycobacterial clearance in mice.     

BAFF Receptor mAb Treatment Ameliorates Development and Progression of Atherosclerosis in Hyperlipidemic ApoE?/? Mice

Aims

Option to attenuate atherosclerosis by depleting B2 cells is currently limited to anti-CD20 antibodies which deplete all B-cell subtypes. In the present study we evaluated the capacity of a monoclonal antibody to B cell activating factor-receptor (BAFFR) to selectively deplete atherogenic B2 cells to prevent both development and progression of atherosclerosis in the ApoE^−/− mouse.

Methods and Results

To determine whether the BAFFR antibody prevents atherosclerosis development, we treated ApoE^−/− mice with the antibody while feeding them a high fat diet (HFD) for 8 weeks. Mature CD93⁻ CD19⁺ B2 cells were reduced by treatment, spleen B-cell zones disrupted and spleen CD20 mRNA expression decreased while B1a cells and non-B cells were spared. Atherosclerosis was ameliorated in the hyperlipidemic mice and CD19⁺ B cells, CD4⁺ and CD8⁺ T cells were reduced in atherosclerotic lesions. Expressions of proinflammatory cytokines, IL1β, TNFα, and IFNγ in the lesions were also reduced, while MCP1, MIF and VCAM-1 expressions were unaffected. Plasma immunoglobulins were reduced, but MDA-oxLDL specific antibodies were unaffected. To determine whether anti-BAFFR antibody ameliorates progression of atherosclerosis, we first fed ApoE^−/− mice a HFD for 6 weeks, and then instigated anti-BAFFR antibody treatment for a further 6 week-HFD. CD93⁻ CD19⁺ B2 cells were selectively decreased and atherosclerotic lesions were reduced by this treatment.

Conclusion

Anti-BAFFR monoclonal antibody selectively depletes mature B2 cells while sparing B1a cells, disrupts spleen B-cell zones and ameliorates atherosclerosis development and progression in hyperlipidemic ApoE^−/− mice. Our findings have potential for clinical translation to manage atherosclerosis-based cardiovascular diseases.     

BAFF promotes Th17 cells and aggravates experimental autoimmune encephalomyelitis

Background

BAFF, in addition to promoting B cell survival and differentiation, may affect T cells. The objective of this study was to determine the effect of BAFF on Th17 cell generation and its ramifications for the Th17 cell-driven disease, EAE.

Methodology/Principal Findings

Th17 cells were increased in BAFF-Tg B6 (B6.BTg) mice and decreased in B6.Baff^−/− mice. Th17 cells in B6.Baff^−/− mice bearing a BAFF Tg (B6.Baff^−/−.BTg mice) were identical to those in B6.BTg mice, indicating that membrane BAFF is dispensable for Th17 cell generation as long as soluble BAFF is plentiful. In T + non-T cell criss-cross co-cultures, Th17 cell generation was greatest in cultures containing B6.BTg T cells and lowest in cultures containing B6.Baff^−/− T cells, regardless of the source of non-T cells. In cultures containing only T cells, Th17 cell generation followed an identical pattern. CD4⁺ cell expression of CD126 (IL-6R α chain) was increased in B6.BTg mice and decreased in B6.Baff^−/− mice, and activation of STAT3 following stimulation with IL-6 + TGF-β was also greatest in B6.BTg cells and lowest in B6.Baff^−/− cells. EAE was clinically and pathologically most severe in B6.BTg mice and least severe in B6.Baff^−/− mice and correlated with MOG_35–55 peptide-induced Th17 cell responses.

Conclusions/Significance

Collectively, these findings document a contribution of BAFF to pathogenic Th17 cell responses and suggest that BAFF antagonism may be efficacious in Th17 cell-driven diseases.     

Intranodal interaction with dendritic cells dynamically regulates surface expression of the co-stimulatory receptor CD226 protein on murine T cells

Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system. Depending on their maturation status, they prime T cells to induce adaptive immunity or tolerance. DCs express CD155, an immunoglobulin-like receptor binding CD226 present on T and natural killer (NK) cells. CD226 represents an important co-stimulator during T cell priming but also serves as an activating receptor on cytotoxic T and NK cells. Here, we report that cells of the T and NK cell lineage of CD155^−/− mice express markedly elevated protein levels of CD226 compared with wild type (WT). On heterozygous CD155^+/− T cells, CD226 up-regulation is half-maximal, implying an inverse gene-dosis effect. Moreover, CD226 up-regulation is independent of antigen-driven activation because it occurs already in thymocytes and naïve peripheral T cells. In vivo, neutralizing anti-CD155 antibody elicits up-regulation of CD226 on T cells demonstrating, that the observed modulation can be triggered by interrupting CD155-CD226 contacts. Adoptive transfers of WT or CD155^−/− T cells into CD155^−/− or WT recipients, respectively, revealed that CD226 modulation is accomplished in trans. Analysis of bone marrow chimeras showed that regulators in trans are of hematopoietic origin. We demonstrate that DCs are capable of manipulating CD226 levels on T cells in vivo but not in vitro, suggesting that the process of T cells actively scanning antigen-presenting DCs inside secondary lymphoid organs is required for CD226 modulation. Hence, a CD226 level divergent from WT may be exploited as a sensor to detect abnormal DC/T-cell cross-talk as illustrated for T cells in mice lacking CCR7.     

LRP5 deficiency down‐regulates Wnt signalling and promotes aortic lipid infiltration in hypercholesterolaemic mice

Low-density lipoprotein receptor-related protein 5 (LRP5) is a member of the LDLR family that orchestrates cholesterol homoeostasis. The role of LRP5 and the canonical Wnt pathway in the vascular wall of dyslipidaemic animals remains unknown. In this study, we analysed the role of LRP5 and the Wnt signalling pathway in mice fed a hypercholesterolaemic diet (HC) to trigger dyslipidaemia. We show that Lrp5^−/− mice had larger aortic lipid infiltrations than wild-type mice, indicating a protective role for LRP5 in the vascular wall. Three members of the LDLR family, Lrp1, Vldlr and Lrp6, showed up-regulated gene expression levels in aortas of Lrp5^−/− mice fed a hypercholesterolaemic diet. HC feeding in Lrp5^−/− mice induced higher macrophage infiltration in the aortas and accumulation of inflammatory cytokines in blood. Wnt/β-CATENIN signalling proteins were down-regulated in HC Lrp5^−/− mice indicating that LRP5 regulates the activation of Wnt signalling in the vascular wall. In conclusion, our findings show that LRP5 and the canonical Wnt pathway down-regulation regulate the dyslipidaemic profile by promoting lipid and macrophage retention in the vessel wall and increasing leucocyte-driven systemic inflammation.     

Murine melanoma-infiltrating dendritic cells are defective in antigen presenting function regardless of the presence of CD4CD25 regulatory T cells

Tumor-infiltrating dendritic cells are often ineffective at presenting tumor-derived antigen in vivo, a defect usually ascribed to the suppressive tumor environment. We investigated the effects of depleting CD4⁺CD25⁺ “natural” regulatory T cells (Treg) on the frequency, phenotype and function of total dendritic cell populations in B16.OVA tumors and in tumor-draining lymph nodes. Intraperitoneal injection of the anti-CD25 monoclonal antibody PC61 reduced Treg frequency in blood and tumors, but did not affect the frequency of tumor-infiltrating dendritic cells, or their expression of CD40, CD86 and MHCII. Tumor-infiltrating dendritic cells from PC61-treated or untreated mice induced the proliferation of allogeneic T cells in vitro, but could not induce proliferation of OVA-specific OTI and OTII T cells unless specific peptide antigen was added in culture. Some proliferation of naïve, OVA-specific OTI T cells, but not OTII T cells, was observed in the tumor-draining LN of mice carrying B16.OVA tumors, however, this was not improved by PC61 treatment. Experiments using RAG1^−/− hosts adoptively transferred with OTI and CD25-depleted OTII cells also failed to show improved OTI and OTII T cell proliferation in vivo compared to C57BL/6 hosts. We conclude that the defective presentation of B16.OVA tumor antigen by tumor-infiltrating dendritic cells and in the tumor-draining lymph node is not due to the presence of “natural” CD4⁺CD25⁺ Treg.     

IL-27 Receptor Signalling Restricts the Formation of Pathogenic,Terminally Differentiated Th1 Cells during Malaria Infection by Repressing IL-12 Dependent Signals

The IL-27R, WSX-1, is required to limit IFN-γ production by effector CD4⁺ T cells in a number of different inflammatory conditions but the molecular basis of WSX-1-mediated regulation of Th1 responses in vivo during infection has not been investigated in detail. In this study we demonstrate that WSX-1 signalling suppresses the development of pathogenic, terminally differentiated (KLRG-1⁺) Th1 cells during malaria infection and establishes a restrictive threshold to constrain the emergent Th1 response. Importantly, we show that WSX-1 regulates cell-intrinsic responsiveness to IL-12 and IL-2, but the fate of the effector CD4⁺ T cell pool during malaria infection is controlled primarily through IL-12 dependent signals. Finally, we show that WSX-1 regulates Th1 cell terminal differentiation during malaria infection through IL-10 and Foxp3 independent mechanisms; the kinetics and magnitude of the Th1 response, and the degree of Th1 cell terminal differentiation, were comparable in WT, IL-10R1^−/− and IL-10^−/− mice and the numbers and phenotype of Foxp3⁺ cells were largely unaltered in WSX-1^−/− mice during infection. As expected, depletion of Foxp3⁺ cells did not enhance Th1 cell polarisation or terminal differentiation during malaria infection. Our results significantly expand our understanding of how IL-27 regulates Th1 responses in vivo during inflammatory conditions and establishes WSX-1 as a critical and non-redundant regulator of the emergent Th1 effector response during malaria infection.     

SUMO2 is essential while SUMO3 is dispensable for mouse embryonic development

Small ubiquitin-like modifier (SUMO1–3) conjugation plays a critical role in embryogenesis. Embryos deficient in the SUMO-conjugating enzyme Ubc9 die at the early postimplantation stage. Sumo1^−/− mice are viable, as SUMO2/3 can compensate for most SUMO1 functions. To uncover the role of SUMO2/3 in embryogenesis, we generated Sumo2- and Sumo3-null mutant mice. Here, we report that Sumo3^−/− mice were viable, while Sumo2^−/− embryos exhibited severe developmental delay and died at approximately embryonic day 10.5 (E10.5). We also provide evidence that SUMO2 is the predominantly expressed SUMO isoform. Furthermore, although Sumo2^+/− and Sumo2^+/−;Sumo3^+/− mice lacked any overt phenotype, only 2 Sumo2^+/−;Sumo3^−/− mice were found at birth in 35 litters after crossing Sumo2^+/−;Sumo3^+/− with Sumo3^−/− mice, and these rare mice were considerably smaller than littermates of the other genotypes. Thus, our findings suggest that expression levels and not functional differences between SUMO2 and SUMO3 are critical for normal embryogenesis.     

The obesity and fatty liver are reduced by plant-derived Pediococcus pentosaceus LP28 in high fat diet-induced obese mice

We evaluated the effect of an oral administration of a plant-derived lactic acid bacterium, Pediococcus pentosaceus LP28 (LP28), on metabolic syndrome by using high fat diet-induced obese mice. The obese mice were divided into 2 groups and fed either a high fat or regular diet for 8 weeks. Each group was further divided into 3 groups, which took LP28, another plant-derived Lactobacillus plantarum SN13T (SN13T) or no lactic acid bacteria (LAB). The lean control mice were fed a regular diet without inducing obesity prior to the experiment. LP28 reduced body weight gain and liver lipid contents (triglyceride and cholesterol), in mice fed a high fat diet for 8 weeks (40%, 54%, and 70% less than those of the control group without LAB, and P = 0.018, P<0.001, and P = 0.021, respectively), whereas SN13T and the heat treated LP28 at 121°C for 15 min were ineffective. Abdominal visceral fat in the high fat diet mice fed with LP28 was also lower than that without LAB by 44%, although it was not significant but borderline (P = 0.076). The sizes of the adipocytes and the lipid droplets in the livers were obviously decreased. A real-time PCR analyses showed that lipid metabolism-related genes, such as CD36 (P = 0.013), SCD1 encoding stearoyl-CoA desaturase 1 (not significant but borderline, P = 0.066), and PPARγ encoding peroxisome proliferator-activated receptor gamma (P = 0.039), were down-regulated by taking LP28 continuously, when compared with those of the control group. In conclusion, LP28 may be a useful LAB strain for the prevention and reduction of the metabolic syndrome.