Macrophages undergo extensive metabolic rewiring upon activation which assist the cell in roles beyond energy production and synthesis of anabolic building blocks. So-called immunometabolites that accumulate upon immune activation can serve as co-factors for enzymes and can act as signaling molecules to modulate cellular processes. As such, the Krebs-cycle-associated metabolites succinate, itaconate and alpha-ketoglutarate (αKG) have emerged as key regulators of macrophage function. Here, we describe that 2-hydroxyglutarate (2HG), which is structurally similar to αKG and exists as two enantiomers, accumulates during later stages of LPS-induced inflammatory responses in mouse and human macrophages. D-2HG was the most abundant enantiomer in macrophages and its LPS-induced accumulation followed the induction of Hydroxyacid-Oxoacid Transhydrogenase (HOT). HOT interconverts αKG and gamma-hydroxybutyrate into D-2HG and succinic semialdehyde, and we here identified this enzyme as being immune-responsive and regulated during the course of macrophage activation. The buildup of D-2HG may be further explained by reduced expression of D-2HG Dehydrogenase (D2HGDH), which converts D-2HG back into αKG, and showed inverse kinetics with HOT and D-2HG levels. We tested the immunomodulatory effects of D-2HG during LPS-induced inflammatory responses by transcriptomic analyses and functional profiling of D-2HG-pre-treated macrophages in vitro and mice in vivo. Together, these data suggest a role for D-2HG in the negative feedback regulation of inflammatory signaling during late-stage LPS-responses in vitro and as a regulator of local and systemic inflammatory responses in vivo. Finally, we show that D-2HG likely exerts distinct anti-inflammatory effects, which are in part independent of αKG-dependent dioxygenase inhibition. Together, this study reveals an immunometabolic circuit resulting in the accumulation of the immunomodulatory metabolite D-2HG that can inhibit inflammatory macrophage responses.

Macrophages are highly plastic, key regulators of inflammation. Deregulation of macrophage activation can lead to excessive inflammation as seen in inflammatory disorders like atherosclerosis, obesity, multiple sclerosis and sepsis. Targeting intracellular metabolism is considered as an approach to reshape deranged macrophage activation and to dampen the progression of inflammatory disorders. ATP citrate lyase (Acly) is a key metabolic enzyme and an important regulator of macrophage activation. Using a macrophage-specific Acly-deficient mouse model, we investigated the role of Acly in macrophages during acute and chronic inflammatory disorders. First, we performed RNA sequencing to demonstrate that Acly-deficient macrophages showed hyperinflammatory gene signatures in response to acute LPS stimulation in vitro . Next, we assessed endotoxin-induced peritonitis in myeloid-specific Acly-deficient mice and show that, apart from increased splenic Il6 expression, systemic and local inflammation were not affected by Acly deficiency. Also during obesity, both chronic low-grade inflammation and whole-body metabolic homeostasis remained largely unaltered in mice with Acly-deficient myeloid cells. Lastly, we show that macrophage-specific Acly deletion did not affect the severity of experimental autoimmune encephalomyelitis (EAE), an experimental model of multiple sclerosis. These results indicate that, despite increasing inflammatory responses in vitro , macrophage Acly deficiency does not worsen acute and chronic inflammatory responses in vivo. Collectively, our results indicate that caution is warranted in prospective long-term treatments of inflammatory disorders with macrophage-specific Acly inhibitors. Together with our earlier observation that myeloid Acly deletion stabilizes atherosclerotic lesions, our findings highlight that therapeutic targeting of macrophage Acly can be beneficial in some, but not all, inflammatory disorders.

Within lymph nodes (LNs), T follicular helper (TFH) cells help B cells to produce antibodies, which can either be protective or autoreactive. Here, we demonstrate that murine LN stromal cells (LNSCs) suppress the formation of autoreactive TFH cells in an antigen-specific manner, thereby significantly reducing germinal center B cell responses directed against the same self-antigen. Mechanistically, LNSCs express and present self-antigens in major histocompatibility complex (MHC) class II, leading to the conversion of naive CD4+ T cells into T regulatory (TREG) cells in an interleukin-2 (IL-2)-dependent manner. Upon blockade of TREG cells, using neutralizing IL-2 antibodies, autoreactive TFH cells are allowed to develop. We conclude that the continuous presentation of self-antigens by LNSCs is critical to generate antigen-specific TREG cells, thereby repressing the formation of TFH cells and germinal center B cell responses. Our findings uncover the ability of LNSCs to suppress the early activation of autoreactive immune cells and maintain peripheral tolerance.

BACKGROUND:Long-term exposure of conventional peritoneal dialysis (PD) fluid is associated with structural membrane alterations and technique failure. Previously, it has been shown that infiltrating IL-17-secreting CD4+T cells and pro-fibrotic M2 macrophages play a critical role in the PD-induced pathogenesis. Although more biocompatible PD solutions are recognized to better preserve the peritoneal membrane integrity, the impact of these fluids on the composition of the peritoneal cell infiltrate is unknown.MATERIALS AND METHODS:In a uremic PD mouse model, we compared the effects of daily instillation of standard lactate (LS) or bicarbonate/lactate-buffered solutions (BLS) and respective controls on peritoneal fibrosis, vascularisation, and inflammation.RESULTS:Daily exposure of LS fluid during a period of 8 weeks resulted in a peritoneal increase of αSMA and collagen accompanied with new vessel formation compared to the BLS group. Effluent from LS-treated mouse showed a higher percentage of CD4+ IL-17+ cell population while BLS exposure resulted in an increased macrophage population. Significantly enhanced inflammatory cytokines such as TGFβ1, TNFα, INFγ, and MIP-1β were detected in the effluent of BLS-exposed mice when compared to other groups. Further, immunohistochemistry of macrophage subset infiltrates in the BLS group confirmed a higher ratio of pro-inflammatory M1 macrophages over the pro-fibrotic M2 subset compared to LS.CONCLUSION:Development of the peritoneal fibrosis and angiogenesis was prevented in the BLS-exposed mice, which may underlie its improved biocompatibility. Peritoneal recruitment of M1 macrophages and lower number of CD4+ IL-17+ cells might explain the peritoneal integrity preservation observed in BLS-exposed mouse.

Peritoneal dialysis (PD) can result in chronic inflammation and progressive peritoneal membrane damage. Alanyl-Glutamine (Ala-Gln), a dipeptide with immunomodulatory effects, improved resistance of mesothelial cells to PD fluids. Recently, interleukin-17 (IL-17) was found to be associated with PD-induced peritoneal damage. Here we studied the capacity of intraperitoneal Ala-Gln administration to protect against peritoneal damage by modulating IL-17 expression in uremic rat and mouse PD exposure models. Supplementation of PD fluid with Ala-Gln resulted in reduced peritoneal thickness, alpha SMA expression and angiogenesis. Addition of Ala-Gln also attenuated the IL-17 pathway expression induced by PD, reflected by substantial reduction or normalization of peritoneal levels of IL-17, transforming growth factor beta, IL-6, and the transcription factor retinoic acid receptor-related orphan receptor gamma T. Moreover, increased levels of IL-17 were associated with PD-induced peritoneal thickening. Conversely, Ala-Gln treatment prevented peritoneal extracellular matrix deposition, an effect seen with IL-17 blockade. Thus, intraperitoneal administration of Ala-Gln, a stable dipeptide commonly used in parenteral nutrition, ameliorates PD-induced peritoneal damage in animal models, in part by modulating IL-17 expression. Hence, Ala-Gln supplementation of dialysate may be a potential strategy to ameliorate peritoneal deterioration during PD.

Different animal models for peritoneal dialysis (PD) have been used in the past decades to develop PD fluids compatible with patient life and to identify markers of peritoneal fibrosis and inflammation. Only few of those studies have taken into account the importance of uraemia-induced alterations at both systemic and peritoneal levels. Moreover, some animal studies which have reported about PD in a uremic setting did not always entirely succeed in terms of uraemia establishment and animal survival. In the present study we induced uraemia in the recently established mouse PD exposure model in order to obtain a more clinically relevant mouse model for kidney patients. This new designed model reflected both the slight thickening of peritoneal membrane induced by uraemia and the significant extracellular matrix deposition due to daily PD fluid instillation. In addition the model offers the opportunity to perform long-term exposure to PD fluids, as it is observed in the clinical setting, and gives the advantage to knock out candidate markers for driving peritoneal inflammatory mechanisms.

Vitamin D deficiency is associated with a range of clinical disorders. To study the mechanisms involved and improve treatments, animal models are tremendously useful. Current vitamin D deficient rat models have important practical limitations, including time requirements when using, exclusively, a vitamin D deficient diet. More importantly, induction of hypovitaminosis D causes significant fluctuations in parathyroid hormone (PTH) and mineral levels, complicating the interpretation of study results. To overcome these shortcomings, we report the successful induction of vitamin D deficiency within three weeks, with stable serum PTH and minerals levels, in Wistar rats. We incorporated two additional manoeuvres compared to a conventional diet. Firstly, the vitamin D depleted diet is calcium (Ca) enriched, to attenuate the development of secondary hyperparathyroidism. Secondly, six intraperitoneal injections of paricalcitol during the first two weeks are given to induce the rapid degradation of circulating vitamin D metabolites. After three weeks, serum 25-hydroxyvitamin D3 (25D) and 1,25-dihydroxyvitamin D3 (1,25D) levels had dropped below detection limits, with unchanged serum PTH, Ca, and phosphate (P) levels. Therefore, this model provides a useful tool to examine the sole effect of hypovitaminosis D, in a wide range of research settings, without confounding changes in PTH, Ca, and P.

Peritoneal dialysis (PD) is associated with structural and functional alterations of the peritoneal membrane, consisting of fibrosis, angiogenesis, and loss of ultrafiltration capacity. Vitamin D receptor activation (VDRA) plays an important role in mineral metabolism and inflammation, but also antiangiogenic and antifibrotic properties have been reported. Therefore, the effects of active vitamin D treatment on peritoneal function and remodeling were investigated. Rats were either kept naïve to PDF exposure or daily exposed to 10 mL PDF and were treated for five or seven weeks with oral paricalcitol or vehicle control. Non-PDF-exposed rats showed no peritoneal changes upon paricalcitol treatment. Paricalcitol reduced endogenous calcitriol but did not affect mineral homeostasis. However, upon PDF exposure, loss of ultrafiltration capacity ensued which was fully rescued by paricalcitol treatment. Furthermore, PD-induced ECM thickening was significantly reduced and omental PD-induced angiogenesis was less pronounced upon paricalcitol treatment. No effect of paricalcitol treatment on total amount of peritoneal cells, peritoneal leukocyte composition, and epithelial to mesenchymal transition (EMT) was observed. Our data indicates that oral VDRA reduces tissue remodeling during chronic experimental PD and prevents loss of ultrafiltration capacity. Therefore, VDRA is potentially relevant in the prevention of treatment technique failure in PD patients.

Peritoneal dialysis (PD)-induced peritonitis leads to dysfunction of the peritoneal membrane. During peritonitis, neutrophils are recruited to the inflammation site by rolling along the endothelium, adhesion, and transmigration through vessel walls. In a rat PD-model, long-term effects of PD-fluids (PDF) on leukocyte-endothelium interactions and neutrophil migration were studied under baseline and inflammatory conditions. Rats received daily conventional-lactate-buffered PDF (Dianeal), bicarbonate/lactate-buffered PDF (Physioneal) or bicarbonate/lactate buffer (Buffer) during five weeks. Untreated rats served as control. Baseline leukocyte rolling and N-formylmethionyl-leucyl-phenylalanine (fMLP) induced levels of transmigration in the mesentery were evaluated and quantified by intra-vital videomicroscopy and immunohistochemistry. Baseline leukocyte rolling was unaffected by buffer treatment, approximately 2-fold increased after Physioneal and 4-7-fold after Dianeal treatment. After starting fMLP superfusion, transmigrated leukocytes appeared outside the venules firstly after Dianeal treatment (15 minutes), thereafter in Physioneal and Buffer groups (20-22 minutes), and finally in control rats (>25 minutes). Newly formed vessels and total number of transmigrated neutrophils were highest in Dianeal-treated animals, followed by Physioneal and Buffer, and lowest in control rats and correlated for all groups to baseline leukocyte rolling (r = 0.78, P < 0.003). This study indicates that the start of inflammatory neutrophil transmigration is related to PDF bio(in)compatibility, whereas over time neutrophil transmigration is determined by the degree of neo-angiogenesis.

Background: Peritoneal dialysis (PD) is associated with functional and structural alterations of the peritoneal membrane, particularly new vessel formation and fibrosis. In addition to anticoagulant effects, heparin displays antiinflammatory and angiostatic properties. Therefore, the effects of administration of heparins on function and morphology of the peritoneal membrane were studied in a rat PD model.Methods: Rats received 10 mL conventional PD fluid (PDF) daily, with or without the addition of unfractionated heparin (UFH) or low molecular weight heparin (LMWH) in the PDF (1 mg/10 mL intraperitoneally) via a mini access port. Untreated rats served as controls. After 5 weeks, a 90-minute functional peritoneal transport test was performed and tissues and peritoneal leukocytes were taken.Results: PD treatment induced loss of ultrafiltration (p < 0.01), a twofold increase in glucose absorption (p < 0.03), increased urea transport (p < 0.02), and loss of sodium sieving (p < 0.03), which were also found in the PDF + heparin groups. Increased peritoneal cell influx and hyaluronan production (p < 0.02) as well as an exchange of mast cells and eosinophils for neutrophils after PD treatment were observed in PD rats; addition of heparin did not affect those changes. Mesothelial regeneration, submesothelial blood vessel and matrix formation, and accumulation of tissue macrophages were seen in PD animals. Spindle-shaped vimentin-positive and cytokeratin-negative cells indicated either partial injury and denudation of mesothelial cells or epithelial-to-mesenchymal transition. Neither UFH nor LMWH affected any of these morphological changes.Conclusion: Within 5 weeks, PD treatment induces a chronic inflammatory condition in the peritoneum, evidenced by high transport, leukocyte recruitment, tissue remodeling, and induction of spindle-shaped cells in the mesothelium. Addition of LMWH or UFH to the PDF did not prevent these adverse PDF-induced peritoneal changes.

BACKGROUND:Daily peritoneal exposure to peritoneal dialysis fluid (PDF) induces severe morphological alterations including fibrosis and angiogenesis that lead to a loss of peritoneal ultrafiltration (UF) capacity. Since cyclooxygenase (COX)-2 is involved in fibrosis and angiogenesis, we investigated the in vivo effects of a selective COX-2 inhibitor (celecoxib) in a rat-PD model.METHODS:Sixteen rats daily received 10 ml of conventional PDF for 4-5 weeks intraperitoneally. Half of them (n = 8) daily received celecoxib (20 mg/kg BW) via oral gavage, and the other half (n = 8) received vehicle via oral gavage. The study also included two control groups (no PDF instillations), each consisting of n = 8 animals that daily received celecoxib or vehicle, respectively, via oral gavage. Functional, morphological and cellular parameters were analysed.RESULTS:PDF exposure induced an inflammatory condition evidenced by the increased leucocyte number and synthesis of MCP-1, VEGF and hyaluronic acid. After PDF exposure, the omentum showed intense angiogenesis and milky spots formation. Parietal peritoneum showed increased angiogenesis, lymphangiogenesis, submesothelial matrix thickness and enhanced expression of mesothelial aquaporin1 (Aqp1). Concomitant PDF and celecoxib exposure drastically reduced PGE2 levels, angiogenesis, lymphangiogenesis, fibrosis and milky spot formation in studied tissues, but did not modify mesothelial Aqp1 expression nor the tissue expression of VEGF and inflammatory markers. PDF exposure induced severe UF failure that celecoxib treatment completely prevented.CONCLUSIONS:Altogether, celecoxib treatment improves UF capacity and reduces morphological alterations in our rat PD model.

Leukocyte infiltration into inflamed tissues is considered to involve sequential steps of rolling over the endothelium, adhesion, and transmigration. In this model, the leukocyte adhesion molecule L-selectin and its ligands expressed on inflamed endothelial cells are involved in leukocyte rolling. We show that upon experimental and human renal ischemia/reperfusion, associated with severe endothelial damage, microvascular basement membrane (BM) heparan sulfate proteoglycans (HSPGs) are modified to bind L-selectin and monocyte chemoattractant protein-1. In an in vitro rolling and adhesion assay, L-selectin-binding HSPGs in artificial BM induced monocytic cell adhesion under reduced flow. We examined the in vivo relevance of BM HSPGs in renal ischemia/reperfusion using mice mutated for BM HSPGs perlecan (Hspg2(Delta3/Delta3)), collagen type XVIII (Col18a1(-/-)), or both (cross-bred Hspg2(Delta3/Delta3)xCol18a1(-/-)) and found that early monocyte/macrophage influx was impaired in Hspg2(Delta3/Delta3)xCol18a1(-/-) mice. Finally, we confirmed our observations in human renal allograft biopsies, showing that loss of endothelial expression of the extracellular endosulfatase HSulf-1 may be a likely mechanism underlying the induction of L-selectin- and monocyte chemoattractant protein-1-binding HSPGs associated with peritubular capillaries in human renal allograft rejection. Our results provide evidence for the concept that not only endothelial but also (microvascular) BM HSPGs can influence inflammatory responses.

Background In experimental peritoneal dialysis (PD) studies, the occurrence of peritonitis is a confounder in the interpretation of effects of chronic peritoneal exposure to dialysis solutions. Since fluid cannot be drained in most experimental PD models in the rat, it is impossible to diagnose peritonitis based on dialysate white blood cell counts. To study the value of serum markers for the presence of peritonitis, alpha-2-macroglobulin (α2M) and albumin were measured in rats with and without peritonitis after chronic exposure to dialysis solutions. To further investigate the time course of these markers in relation to the severity of peritonitis, nondialyzed rats were challenged with increasing numbers of bacteria and followed for 28 days. Methods In the first study, α2M and albumin were measured in rats exposed to glucose/lactate-based dialysis fluid before sacrifice. A comparison was made between animals with peritonitis, as judged from the presence of extensive infiltrates after sacrifice (gold standard) and/or clinical signs of peritonitis, or absence of peritonitis and infiltrates. In the second study, rats were intraperitoneally (IP) injected with 3 different concentrations of Staphylococcus aureus, and serum α2M and albumin were measured at various time points. Results In the first study, serum α2M was higher and serum albumin was lower in animals with peritonitis compared to animals without peritonitis (both p < 0.05). In the second study, induction of α2M was clearly dependent on the inoculum concentration. Peak values of α2M were found at days 1 and 3. At all time points after inoculation, α2M was higher in all injected groups compared to the control group. Serum albumin values decreased in the highest inoculum group and remained decreased until 28 days after IP injection. Despite a low sensitivity, serum α2M >40 mg/L and albumin <32 g/L had a specificity of 100% for peritonitis. Conclusions Measurement of α2M and albumin once per month is an additional tool in the diagnosis of silent peritonitis in the chronic peritoneal exposure model in the rat. Levels of α2M >40 mg/L and albumin <32 g/L are strong indicators for peritonitis. However, normal values do not exclude infectious peritonitis.

Patients treated with peritoneal dialysis (PD) are at risk for development of ultrafiltration failure and peritonitis. The relative unphysiologic composition of the currently used peritoneal dialysis fluids (PDF) is a major cause for the development of morphologic changes of the peritoneal membrane such as fibrosis and new vessel formation, ultimately resulting in ultrafiltration failure. In recent years, a major research focus has become the development of new and improved PDF. Typically, the first phase of biocompatibility testing of new PDF involves in vitro testing, using cell culture systems such as primary mesothelial cells or peritoneal macrophages. In vivo studies using animal models permit the analysis of biocompatibility under conditions that allow for cell-to-cell interactions and dynamic changes in solution composition that more closely mimic the clinical situation. In this paper, we will review the applicability of a peritoneal exposure model in the rat to study PDF biocompatibility-related issues.

Because of its dynamic structure, the omentum plays a key role in the immunity of the peritoneal cavity by orchestrating peritoneal cell recruitment. Because mast cells accumulate in the omentum upon experimental peritoneal dialysis (PD) and may produce angiogenic/profibrotic factors, it was hypothesized that mast cells mediate omental tissue remodeling during PD. Daily treatment with conventional PD fluid (PDF) for 5 wk resulted in a strong omental remodeling response, characterized by an approximately 10-fold increase in mast cell density (P < 0.01), an approximately 20-fold increase in vessel density (P < 0.02), an approximately 20-fold increase in the number of milky spots (P < 0.01), and a four-fold increase in submesothelial matrix thickness (P < 0.0003) in wild-type rats. In contrast, all PDF-induced omental changes were significantly reduced in mast cell-deficient Ws/Ws rats or in wild-type rats that were treated orally with a mast cell stabilizer cromoglycate. A time-course experiment showed mast cell accumulation immediately before the formation of blood vessels and milky spots. Functionally, PDF evoked a peritoneal cell influx, which was significantly reduced in Ws/Ws rats (P < 0.04) and in wild-type rats that were treated with cromoglycate (P < 0.03). Cromoglycate treatment also completely prevented PDF-induced omental adhesions to the catheter tip (P = 0.0002). Mesothelial damage, angiogenesis, and fibrosis of mesentery and parietal peritoneum as well as glucose absorption rate and ultrafiltration capacity proved to be mast cell independent. Data strongly support the hypothesis that mast cells mediate PDF-induced omental tissue remodeling and, subsequently, peritoneal cell influx and adhesion formation, providing therapeutic possibilities of modulating omental function.

BACKGROUND Chronic exposure to conventional peritoneal dialysis fluid (PDF) is associated with functional and structural alterations of the peritoneal membrane. The bioincompatibility of conventional PDF can be due to hypertonicity, high glucose concentration, lactate buffering system, presence of glucose degradation products (GDPs) and/or acidic pH. Although various investigators have studied the sole effects of hyperosmolarity, high glucose, GDPs and lactate buffer in experimental PD, less attention has been paid to the chronic impact of low pH in vivo. METHODS Rats received daily 10 ml of either conventional lactate-buffered PDF (pH 5.2; n=7), a standard bicarbonate/lactate-buffered PDF with physiological pH (n=8), bicarbonate/lactate-buffered PDF with acidic pH (adjusted to pH 5.2 with 1 N hydrochloride, n=5), or bicarbonate/lactate buffer, without glucose, pH 7.4 (n=7). Fluids were instilled via peritoneal catheters connected to implanted subcutaneous mini vascular access ports for 8 weeks. Control animals with or without peritoneal catheters served as control groups (n=8/group). Various functional (2 h PET) and morphological/cellular parameters were analyzed. RESULTS Compared with control groups and the buffer group, conventional lactate-buffered PDF induced a number of morphological/cellular changes, including angiogenesis and fibrosis in various peritoneal tissues (all parameters P<0.05), accompanied by increased glucose absorption and reduced ultrafiltration capacity. Daily exposure to standard or acidified bicarbonate/lactate-buffered PDF improved the performance of the peritoneal membrane, evidenced by reduced new vessel formation in omentum (P<0.02) and parietal peritoneum (P<0.008), reduced fibrosis (P<0.02) and improved ultrafiltration capacity. No significant differences were found between standard and acidified bicarbonate/lactate-buffered PDF. During PET, acidic PDF was neutralized within 15 to 20 min. CONCLUSION The bicarbonate/lactate-buffered PDF, acidity per se did not contribute substantially to peritoneal worsening in our in vivo model for PD, which might be explained by the buffering capacity of the peritoneum.