Obesity Impairs Lymphatic Fluid Transport and Dendritic Cell Migration to Lymph Nodes

Introduction

Obesity is a major cause of morbidity and mortality resulting in pathologic changes in virtually every organ system. Although the cardiovascular system has been a focus of intense study, the effects of obesity on the lymphatic system remain essentially unknown. The purpose of this study was to identify the pathologic consequences of diet induced obesity (DIO) on the lymphatic system.

Methods

Adult male wild-type or RAG C57B6-6J mice were fed a high fat (60%) or normal chow diet for 8–10 weeks followed by analysis of lymphatic transport capacity. In addition, we assessed migration of dendritic cells (DCs) to local lymph nodes, lymph node architecture, and lymph node cellular make up.

Results

High fat diet resulted in obesity in both wild-type and RAG mice and significantly impaired lymphatic fluid transport and lymph node uptake; interestingly, obese wild-type but not obese RAG mice had significantly impaired migration of DCs to the peripheral lymph nodes. Obesity also resulted in significant changes in the macro and microscopic anatomy of lymph nodes as reflected by a marked decrease in size of inguinal lymph nodes (3.4-fold), decreased number of lymph node lymphatics (1.6-fold), loss of follicular pattern of B cells, and dysregulation of CCL21 expression gradients. Finally, obesity resulted in a significant decrease in the number of lymph node T cells and increased number of B cells and macrophages.

Conclusions

Obesity has significant negative effects on lymphatic transport, DC cell migration, and lymph node architecture. Loss of T and B cell inflammatory reactions does not protect from impaired lymphatic fluid transport but preserves DC migration capacity. Future studies are needed to determine how the interplay between diet, obesity, and the lymphatic system modulate systemic complications of obesity.     

Characterization of the systemic loss of dendritic cells in murine lymph nodes during polymicrobial sepsis

Dendritic cells (DCs) play a key role in critical illness and are depleted in spleens from septic patients and mice. To date, few studies have characterized the systemic effect of sepsis on DC populations in lymphoid tissues. We analyzed the phenotype of DCs and Th cells present in the local (mesenteric) and distant (inguinal and popliteal) lymph nodes of mice with induced polymicrobial sepsis (cecal ligation and puncture). Flow cytometry and immunohistochemical staining demonstrated that there was a significant local (mesenteric nodes) and partial systemic (inguinal, but not popliteal nodes) loss of DCs from lymph nodes in septic mice, and that this process was associated with increased apoptosis. This sepsis-induced loss of DCs occurred after CD3(+)CD4(+) T cell activation and loss in the lymph nodes, and the loss of DCs was not preceded by any sustained increase in their maturation status. In addition, there was no preferential loss of either mature/activated (MHCII(high)/CD86(high)) or immature (MHCII(low)/CD86(low)) DCs during sepsis. However, there was a preferential loss of CD8(+) DCs in the local and distant lymph nodes. The loss of DCs in lymphoid tissue, particularly CD8(+) lymphoid-derived DCs, may contribute to the alterations in acquired immune status that frequently accompany sepsis.     

Diurnal dynamics of spatial-temporal organization of lymph nodes

Experiments, performed on CBA mice demonstrate that all the parameters of lymph nodes of various localization possess a diurnal rhythmicity. Certain lympho-endocrine connections participate in realization of the regional peculiarities of the diurnal rhythms via regulation of redistribution of streams of the migrating lymphoid cells, that form the lymph node structures. There are certain differences in endogenous glucocorticoids action, depending on the level of the motor activity, that determines level of the mast cells activation, value of lymph-formation and blood stream intensity in the drained region. Concrete examples of the diurnal spatial-time organization of functional steps are described in inguinal, mesenteric and bifurcational lymph nodes.     

Topical application of capsaicin reduces visceral adipose fat by affecting adipokine levels in high‐fat diet‐induced obese mice

Objective:

Visceral obesity contributes to the development of obesity‐related disorders such as diabetes, hyperlipidemia, and fatty liver disease, as well as cardiovascular diseases. In this study, we determined whether topical application of capsaicin can reduce fat accumulation in visceral adipose tissues.

Methods and Results:

We first observed that topical application of 0.075% capsaicin to male mice fed a high‐fat diet significantly reduced weight gain and visceral fat. Fat cells were markedly smaller in the mesenteric and epididymal adipose tissues of mice treated with capsaicin cream. The capsaicin treatment also lowered serum levels of fasting glucose, total cholesterol, and triglycerides. Immunoblot analysis and RT‐PCR revealed increased expression of adiponectin and other adipokines including peroxisome proliferator‐activated receptor (PPAR) α, PPARγ, visfatin, and adipsin, but reduced expression of tumor necrosis factor‐α and IL‐6.

Conclusions:

These results indicate that topical application of capsaicin to obese mice limits fat accumulation in adipose tissues and may reduce inflammation and increase insulin sensitivity.     

Atrophy of mesenteric lymph nodes in experimental Chagas' disease: differential role of Fas/Fas-L and TNFRI/TNF pathways

It is currently accepted that experimental acute infection by Trypanosoma cruzi promotes changes in secondary lymphoid organs, with general T and B lymphocyte polyclonal activation. Here we show that mesenteric lymph nodes (MLN) of acutely infected mice show severe atrophy due to extensive lymphocyte apoptosis. Accordingly, clusters of apoptotic cells are detected in the initial phase of infection in MLN but not in subcutaneous nodes. Moreover, such atrophy is independent of the infection route, parasite load or the mouse strain used. Studies in Fas-L deficient (BALB gld/gld+/+) and in TNF type 1 receptor (p55-/-) knockout mice indicate that both molecules are involved in MLN atrophy: Fas-L participates in cell death of CD4+ as well as B lymphocytes, whereas the TNF type 1 receptor is important for the apoptosis of CD4+ and CD8+ T lymphocytes. In contrast, perforin does not play a role, as lymph nodes from perforin-deficient mice do not behave differently from the corresponding wild types. Our data support the concept that, even in a systemic infection, differential (even opposing) responses can be found in different lymph node chains.     

Mice Lacking C1q Are Protected from High Fat Diet-induced Hepatic Insulin Resistance and Impaired Glucose Homeostasis

Complement activation is implicated in the development of obesity and insulin resistance, and loss of signaling by the anaphylatoxin C3a prevents obesity-induced insulin resistance in mice. Here we have identified C1q in the classical pathway as required for activation of complement in response to high fat diets. After 8 weeks of high fat diet, wild-type mice became obese and developed glucose intolerance. This was associated with increased apoptotic cell death and accumulation of complement activation products (C3b/iC3b/C3c) in liver and adipose tissue. Previous studies have shown that high fat diet-induced apoptosis is dependent on Bid; here we report that Bid-mediated apoptosis was required for complement activation in adipose and liver. Although C1qa deficiency had no effect on high fat diet-induced apoptosis, accumulation of complement activation products and the metabolic complications of high fat diet-induced obesity were dependent on C1q. When wild-type mice were fed a high fat diet for only 3 days, hepatic insulin resistance was associated with the accumulation of C3b/iC3b/C3c in the liver. Mice deficient in C3a receptor were protected against this early high fat diet-induced hepatic insulin resistance, whereas mice deficient in the negative complement regulator CD55/DAF were more sensitive to the high fat diet. C1qa^−/− mice were also protected from high fat diet-induced hepatic insulin resistance and complement activation. Evidence of complement activation was also detected in adipose tissue of obese women compared with lean women. Together, these studies reveal an important role for C1q in the classical pathway of complement activation in the development of high fat diet-induced insulin resistance.     

Down-regulation of microsomal prostaglandin E2 synthase-1 in adipose tissue by high-fat feeding

Objective: Prostaglandin (PG)E₂ is a lipid mediator implicated in inflammatory diseases and in the regulation of lipolysis and adipocyte differentiation. This work was, thus, undertaken to study the regulation of the various PGE₂ synthases (PGESs) in obesity. Research Methods and Procedures: C57Bl/6 mice were subjected to a high‐fat or regular diet for 12 weeks. The levels of PGE₂ in white adipose tissue (WAT) of lean and obese mice were quantified by liquid chromatography‐mass spectrometry, and the change in expression of the three major PGES caused by diet‐induced obesity was characterized by Western blotting. Human preadipocytes and 3T3‐L1 cells were used to assess the expression of microsomal prostaglandin E₂ synthase‐1 (mPGES‐1) during adipogenesis. Results: mPGES‐1, mPGES‐2, and cytosolic PGES proteins were all detected in WAT of lean animals. mPGES‐1 was expressed at higher levels in WAT than in any other tissues examined and was more abundant (3‐ to 4‐fold) in epididymal (visceral) compared with inguinal (subcutaneous) WAT. Expression of mPGES‐1 was also detected in undifferentiated and differentiated 3T3‐L1 cells and in human primary subcutaneous preadipocytes at all stages of adipogenesis. The mPGES‐1 protein was substantially down‐regulated in epididymal and inguinal WAT of obese mice, whereas mPGES‐2 and cytosolic PGES remained relatively stable. Concordantly, the PGE₂ levels in obese inguinal WAT were significantly lower than those of lean animals. Discussion: These data suggest that mPGES‐1 is the major form of PGESs contributing to the synthesis of PGE₂ in WAT and that its down‐regulation might be involved in the alterations of lipolysis and adipogenesis associated with obesity.     

MAdCAM-1 expressing sacral lymph node in the lymphotoxin beta-deficient mouse provides a site for immune generation following vaginal herpes simplex virus-2 infection

The members of the lymphotoxin (LT) family of molecules play a critical role in lymphoid organogenesis. Whereas LT alpha-deficient mice lack all lymph nodes and Peyer's patches, mice deficient in LT beta retain mesenteric lymph nodes and cervical lymph nodes, suggesting that an LT beta-independent pathway exists for the generation of mucosal lymph nodes. In this study, we describe the presence of a lymph node in LT beta-deficient mice responsible for draining the genital mucosa. In the majority of LT beta-deficient mice, a lymph node was found near the iliac artery, slightly misplaced from the site of the sacral lymph node in wild-type mice. The sacral lymph node of the LT beta-deficient mice, as well as that of the wild-type mice, expressed the mucosal addressin cell adhesion molecule-1 similar to the mesenteric lymph node. Following intravaginal infection with HSV type 2, activated dendritic cells capable of stimulating a Th1 response were found in this sacral lymph node. Furthermore, normal HSV-2-specific IgG responses were generated in the LT beta-deficient mice following intravaginal HSV-2 infection even in the absence of the spleen. Therefore, an LT beta-independent pathway exists for the development of a lymph node associated with the genital mucosa, and such a lymph node serves to generate potent immune responses against viral challenge.     

Early lymphocytic responses to Heligmosomoides polygyrus infections in mice

Responses to parasite antigens were studied in three strains of mice, BALB/c, CBA and NIH, during the initial phases of a primary infection with the intestinal nematode Heligmosomoides polygyrus. Changes in the rate of in vivo cell division were analysed in mesenteric lymph nodes and spleens during the phases of larval maturation and adult establishment, and related to changes in organ size and cellularity. The nature of the proliferating cell populations was also investigated by flow cytometry, carried out on cell suspensions prepared at the time when larval development was complete. The variation in the ability of the strains of mice to become resistant to a challenge infection was manifest as only slight differences in their initial responses to infection. All three strains showed an increase in 125I-iododeoxyuridine incorporation in their mesenteric lymph nodes and spleen, and an increase in B cell frequency over that of T cells in the draining lymph nodes. Although lymph node weight in NIH mice continued to rise over a 4 week period, the majority of responses measured were short lived, peaking 10 to 14 days after infection. The low responder status of CBA mice was thus reflected in a transient and relatively small enlargement of lymphoid tissues, but their early proliferative responses to antigen were similar in scale to those of responder strains.     

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (Bénézech et al.¹⁴). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP⁺ embryonic adipose tissue and make a LN/ EYFP⁺ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP⁺ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.     

Macrophage-colony stimulating factor in obese adipose tissue: studies with heterozygous op/+ mice

Objective: We examined the gene expression of macrophage‐colony stimulating factor (M‐CSF) in mice with diet‐induced obesity and in genetically obese mice. We also examined the effect of decreased M‐CSF signaling on the susceptibility to obesity and macrophage recruitment into the adipose tissue of mice. Research Methods and Procedures: The adipose tissue from mice with diet‐induced obesity, obese KKA^y mice, and ob/ob obese mice was used for RNA preparation. Production of M‐CSF and monocyte chemoattractant protein‐1 (MCP‐1) was examined by quantitative real‐time polymerase chain reaction (PCR) and enzyme‐linked immunosorbent assay. The op/+ heterozygous mice, with decreased functional M‐CSF expression, were placed on a high‐fat diet or crossed with KKA^y mice to study the susceptibility to obesity. The gene expression of macrophage markers in adipose tissue was examined. Results: The expression of M‐CSF was not significantly changed in mice on a high‐fat diet or in either type of genetic obesity (KKA^y or ob/ob mice). No change in the degree of obesity or macrophage‐related gene expression (F4/80, CD68, and MCP‐1) in the adipose tissue was observed in op/+ mice compared with +/+ control mice, which were either treated with a high‐fat diet or crossed with KKA^y mice. Discussion: This study demonstrated that there was no significant change in the expression of M‐CSF in the adipose tissue from obese mice and only a minor phenotypic change, such as macrophage infiltration, in the adipose tissue from op/+ mice, suggesting that M‐CSF does not play a major role in macrophage recruitment in the adipose tissue of obese mice.     

Regeneration of autotransplanted lymph node fragments

Summary In normal young minipigs thin slices of autologous mesenteric or superficial inguinal lymph nodes were implanted either in the greater omentum or subcutaneously in the groin region. The regeneration was studied histologically and connections between the afferent lymphatics and the regenerated tissue were checked. In the greater omentum, no regenerated lymph node tissue was found. In the inguinal region, lymphoid tissue with all the typical lymph node compartments was identified following antigenic stimulation in the draining area. Sinuses, germinal centres with a lymphatic corona, and a paracortex with typical high endothelial venules were seen. There was evidence of afferent lymphatics, e.g., macroscopically visible lymphatics, the occurrence of a subcutaneously injected dye, the effect of antigenic stimulation and a normal lymph node structure. Avascular transplants of autologous lymph node fragments regenerate subcutaneously, possibly providing a future technique for treating lymphoedema after radical excision or irradiation of lymph nodes.     

Apoptosis of antigen-specific T cells induced by oral administration of antigen: comparison of intestinal and non-intestinal immune organs.

Oral administration of a protein without adjuvant brings about oral tolerance (systemic hyporesponsiveness) to that protein by mechanisms such as antigen-induced apoptosis. We monitored the number and apoptosis induction of CD4+ T cells in antigen-specific T cell receptor transgenic mice fed the antigen ovalbumin to identify where events leading to oral tolerance occurred. The antigen was distributed throughout the body, causing apoptosis and a decrease in cell number of CD4+ T cells in most of the lymphoid system: the spleen, peripheral lymph nodes, and the thymus which was not previously reported to be affected. Although apoptosis was induced in the Peyer's patches, the cell number did not change. Unexpectedly, T cells in the mesenteric lymph nodes did not undergo apoptosis; instead, they were more numerous as compared to that in the case of control animals not administered the antigen. The results suggested that the orally administered antigen activated the intestinal immune system, while it induced immune tolerance in other sites.     

Obesity and triple‐negative‐breast‐cancer: Is apelin a new key target?

Epidemiological studies have shown that obese subjects have an increased risk of developing triple‐negative breast cancer (TNBC) and an overall reduced survival. However, the relation between obesity and TNBC remains difficult to understand. We hypothesize that apelin, an adipokine whose levels are increased in obesity, could be a major factor contributing to both tumour growth and metastatization in TNBC obese patients. We observed that development of obesity under high‐fat diet in TNBC tumour‐bearing mice significantly increased tumour growth. By showing no effect of high‐fat diet in obesity‐resistant mice, we demonstrated the necessity to develop obesity‐related disorders to increase tumour growth. Apelin mRNA expression was also increased in the subcutaneous adipose tissue and tumours of obese mice. We further highlighted that the reproduction of obesity‐related levels of apelin in lean mice led to an increased TNBC growth and brain metastases formation. Finally, injections of the apelinergic antagonist F13A to obese mice significantly reduced TNBC growth, suggesting that apelinergic system interference could be an interesting therapeutic strategy in the context of obesity and TNBC.     

Proteasome inhibitor bortezomib ameliorates intestinal injury in mice

Background

Bortezomib is a proteasome inhibitor that has shown impressive efficacy in the treatment of multiple myeloma. In mice, the addition of dextran sulfate sodium (DSS) to drinking water leads to acute colitis that can serve as an experimental animal model for human ulcerative colitis.

Methodology/Principal Findings

Bortezomib treatment was shown to potently inhibit murine DSS-induced colitis. The attenuation of DSS-induced colitis was associated with decreased inflammatory cell infiltration in the colon. Specifically, bortezomib-treated mice showed significantly decreased numbers of CD4⁺ and CD8⁺ T cells in the colon and mesenteric lymph nodes. Bortezomib treatment significantly diminished interferon (IFN)-γ expression in the colon and mesenteric lymph nodes. Furthermore, cytoplasmic IFN-γ production by CD4⁺ and CD8⁺ T cells in mesenteric lymph nodes was substantially decreased by bortezomib treatment. Notably, bortezomib enhanced T cell apoptosis by inhibiting nuclear factor-κB activation during DSS-induced colitis.

Conclusions/Significance

Bortezomib treatment is likely to induce T cell death, thereby suppressing DSS-induced colitis by reducing IFN-γ production.     

In vitro development of a primary antibody response with lymph node cells.

Utilizing a variety of lymphoid tissues from three common laboratory species, comparative studies were performed to investigate the competence of the dissociated cells to respond to a heterologous erythrocyte with the development of specific plaque-forming cells. Dissociated spleen cells harvested from BDF1 mice consistently developed specific plaque-forming cells (PFC) to sheep red blood cells (SRBC), while hamster spleen cells inconsistently developed specific antibody-forming cells to SRBC. Under identical conditions, guinea pig spleen cells did not develop significant numbers of PFC to SRBC. However, lymph node cell cultures of all three species tested yielded specific PFC. In the mouse and hamster lymph node cell cultures, the yield of PFC per culture or per 10⁶ recovered viable cells was always greater than the yield from companion spleen cell cultures. Guinea pig mesenteric lymph node cell cultures developed the major PFC response to SRBC, while both mesenteric and peripheral lymph node cell cultures from hamsters were equivalent in their response to SRBC. The data demonstrate that it is possible to develop a primary antibody response to SRBC in vitro utilizing normal endogenous hamster or guinea pig lymphoid cells, if lymph nodes are the source of cells.     

Further evidence for the existence of B-lymphocyte subpopulations.

We have studied the homing properties of B lymphocytes by using ⁵¹Cr-labeled lymphoid cells obtained from athymic, nu/nu mice, and animals made T-lymphocyte deficient by thymectomy and lethal irradiation followed by reconstitution with syngeneic bone marrow. Comparison was made to the patterns of distribution observed when cell preparations containing normal numbers of T and B lymphocytes were migrated. A small but significant percentage of labeled lymphocytes from lymph nodes, spleen, Peyer's Patches, and bone marrow of T-cell-deficient animals was shown to be lymph node seeking. Secondary transfers of lymph node cells from primary recipients caused enrichment of this lymph node-seeking population. Treatment of T-lymphocyte-deficient lymphoid cell preparations with neuraminidase reduced the percentages of cells homing to the lymph nodes. The data showed that B lymphocytes exhibit unique homing properties when injected into normal recipients. In addition, direct comparison of the homing patterns of B lymphocytes prepared from spleen and lymph nodes of athymic mice revealed differences suggesting that these lymphoid organs contained unique mixtures of at least two different kinds of B cell. The evidence supports the notion that the B-lymphocyte populations contain at least two subpopulations, one of which possesses the ability to home to lymph nodes.