Beta-Glucan from S. cerevisiae Protected AOM-Induced Colon Cancer in cGAS-Deficient Mice Partly through Dectin-1-Manipulated Macrophage Cell Energy.

Although the impacts of Saccharomyces cerevisiae on cancers are mentioned, data on its use in mice with cyclic GMP-AMP synthase deficiency (cGAS-/-) are even rarer. Here, 12 weeks of oral administration of S. cerevisiae protected cGAS-/- mice from azoxymethane (AOM)-induced colon cancers, partly through dysbiosis attenuation (fecal microbiome analysis). In parallel, a daily intralesional injection of a whole glucan particle (WGP; the beta-glucan extracted from S. cerevisiae) attenuated the growth of subcutaneous tumor using MC38 (murine colon cancer cell line) in cGAS-/- mice. Interestingly, the incubation of fluorescent-stained MC38 with several subtypes of macrophages, including M1 (using Lipopolysaccharide; LPS), M2 (IL-4), and tumor-associated macrophages (TAM; using MC38 supernatant activation), could not further reduce the tumor burdens (fluorescent intensity) compared with M0 (control culture media). However, WGP enhanced tumoricidal activities (fluorescent intensity), the genes of M1 pro-inflammatory macrophage polarization (IL-1ß and iNOS), and Dectin-1 expression and increased cell energy status (extracellular flux analysis) in M0, M2, and TAM. In M1, WGP could not increase tumoricidal activities, Dectin-1, and glycolysis activity, despite the upregulated IL-1ß. In conclusion, S. cerevisiae inhibited the growth of colon cancers through dysbiosis attenuation and macrophage energy activation, partly through Dectin-1 stimulation. Our data support the use of S. cerevisiae for colon cancer protection.

Candida Administration in Bilateral Nephrectomy Mice Elevates Serum (1→3)-ß-D-glucan That Enhances Systemic Inflammation Through Energy Augmentation in Macrophages.

Systemic inflammation, from gut translocation of organismal molecules, might worsen uremic complications in acute kidney injury (AKI). The monitoring of gut permeability integrity and/or organismal molecules in AKI might be clinically beneficial. Due to the less prominence of Candida albicans in human intestine compared with mouse gut, C. albicans were orally administered in bilateral nephrectomy (BiN) mice. Gut dysbiosis, using microbiome analysis, and gut permeability defect (gut leakage), which was determined by fluorescein isothiocyanate-dextran and intestinal tight-junction immunofluorescent staining, in mice with BiN-Candida was more severe than BiN without Candida. Additionally, profound gut leakage in BiN-Candida also resulted in gut translocation of lipopolysaccharide (LPS) and (1→3)-ß-D-glucan (BG), the organismal components from gut contents, that induced more severe systemic inflammation than BiN without Candida. The co-presentation of LPS and BG in mouse serum enhanced inflammatory responses. As such, LPS with Whole Glucan Particle (WGP, a representative BG) induced more severe macrophage responses than LPS alone as determined by supernatant cytokines and gene expression of downstream signals (NFκB, Malt-1 and Syk). Meanwhile, WGP alone did not induced the responses. In parallel, WGP (with or without LPS), but not LPS alone, accelerated macrophage ATP production (extracellular flux analysis) through the upregulation of genes in mitochondria and glycolysis pathway (using RNA sequencing analysis), without the induction of cell activities. These data indicated a WGP pre-conditioning effect on cell energy augmentation. In conclusion, Candida in BiN mice accelerated gut translocation of BG that augmented cell energy status and enhanced pro-inflammatory macrophage responses. Hence, gut fungi and BG were associated with the enhanced systemic inflammation in acute uremia.

Lipopolysaccharide-Enhanced Responses against Aryl Hydrocarbon Receptor in FcgRIIb-Deficient Macrophages, a Profound Impact of an Environmental Toxin on a Lupus-Like Mouse Model.

Fc gamma receptor IIb (FcgRIIb) is the only inhibitory-FcgR in the FcgR family, and FcgRIIb-deficient (FcgRIIb-/-) mice develop a lupus-like condition with hyper-responsiveness against several stimulations. The activation of aryl hydrocarbon receptor (Ahr), a cellular environmental sensor, might aggravate activity of the lupus-like condition. As such, 1,4-chrysenequinone (1,4-CQ), an Ahr-activator, alone did not induce supernatant cytokines from macrophages, while the 24 h pre-treatment by lipopolysaccharide (LPS), a representative inflammatory activator, prior to 1,4-CQ activation (LPS/1,4-CQ) predominantly induced macrophage pro-inflammatory responses. Additionally, the responses from FcgRIIb-/- macrophages were more prominent than wild-type (WT) cells as determined by (i) supernatant cytokines (TNF-α, IL-6, and IL-10), (ii) expression of the inflammation associated genes (NF-κB, aryl hydrocarbon receptor, iNOS, IL-1ß and activating-FcgRIV) and cell-surface CD-86 (a biomarker of M1 macrophage polarization), and (iii) cell apoptosis (Annexin V), with the lower inhibitory-FcgRIIb expression. Moreover, 8-week-administration of 1,4-CQ in 8 week old FcgRIIb-/- mice, a genetic-prone lupus-like model, enhanced lupus characteristics as indicated by anti-dsDNA, serum creatinine, proteinuria, endotoxemia, gut-leakage (FITC-dextran), and glomerular immunoglobulin deposition. In conclusion, an Ahr activation worsened the disease severity in FcgRIIb-/- mice possibly through the enhanced inflammatory responses. The deficiency of inhibitory-FcgRIIb in these mice, at least in part, prominently enhanced the pro-inflammatory responses. Our data suggest that patients with lupus might be more vulnerable to environmental pollutants.

Prominent Indomethacin-Induced Enteropathy in Fcgriib Defi-cient lupus Mice: An Impact of Macrophage Responses and Immune Deposition in Gut.

A high dose of NSAIDs, a common analgesic, might induce lupus activity through several NSAIDs adverse effects including gastrointestinal permeability defect (gut leakage) and endotoxemia. Indomethacin (25 mg/day) was orally administered for 7 days in 24-wk-old Fc gamma receptor IIb deficient (FcgRIIb-/-) mice, an asymptomatic lupus model (increased anti-dsDNA without lupus nephritis), and age-matched wild-type (WT) mice. Severity of indomethacin-induced enteropathy in FcgRIIb-/- mice was higher than WT mice as demonstrated by survival analysis, intestinal injury (histology, immune-deposition, and intestinal cytokines), gut leakage (FITC-dextran assay and endotoxemia), serum cytokines, and lupus characteristics (anti-dsDNA, renal injury, and proteinuria). Prominent responses of FcgRIIb-/- macrophages toward lipopolysaccharide (LPS) compared to WT cells due to the expression of only activating-FcgRs without inhibitory-FcgRIIb were demonstrated. Extracellular flux analysis indicated the greater mitochondria activity (increased respiratory capacity and respiratory reserve) in FcgRIIb-/- macrophages with a concordant decrease in glycolysis activity when compared to WT cells. In conclusion, gut leakage-induced endotoxemia is more severe in indomethacin-administered FcgRIIb-/- mice than WT, possibly due to the enhanced indomethacin toxicity from lupus-induced intestinal immune-deposition. Due to a lack of inhibitory-FcgRIIb expression, mitochondrial function, and cytokine production of FcgRIIb-/- macrophages were more prominent than WT cells. Hence, lupus disease-activation from NSAIDs-enteropathy-induced gut leakage is possible.

Endocytosis of red blood cell extracellular vesicles by macrophages leads to cytoplasmic heme release and prevents foam cell formation in atherosclerosis.

Extracellular vesicles (EVs) can be produced from red blood cells (RBCs) on a large scale and used to deliver therapeutic payloads efficiently. However, not much is known about the native biological properties of RBCEVs. Here, we demonstrate that RBCEVs are primarily taken up by macrophages and monocytes. This uptake is an active process, mediated mainly by endocytosis. Incubation of CD14+ monocytes with RBCEVs induces their differentiation into macrophages with an Mheme-like phenotype, characterized by upregulation of heme oxygenase-1 (HO-1) and the ATP-binding cassette transporter ABCG1. Moreover, macrophages that take up RBCEVs exhibit a reduction in surface CD86 and decreased secretion of TNF-α under inflammatory stimulation. The upregulation of HO-1 is attributed to heme derived from haemoglobin in RBCEVs. Heme is released from internalized RBCEVs in late endosomes and lysosomes via the heme transporter, HRG1. Consequently, RBCEVs exhibit the ability to attenuate foam cell formation from oxidized low-density lipoproteins (oxLDL)-treated macrophages in vitro and reduce atherosclerotic lesions in ApoE knockout mice on a high-fat diet. In summary, our study reveals the uptake mechanism of RBCEVs and their delivery of heme to macrophages, suggesting the potential application of RBCEVs in the treatment of atherosclerosis.

Candida Administration Worsens Neutrophil Extracellular Traps in Renal Ischemia Reperfusion Injury Mice: An Impact of Gut Fungi on Acute Kidney Injury.

Because of gut-barrier defect (gut-leakage) after acute kidney injury (AKI) and higher abundance of Candida albicans in human intestines compared with mouse guts, Candida administration in renal ischemia reperfusion injury (I/R) mice possibly more closely resemble patients with AKI than non-Candida model. Fungi in feces were detectable only in mice with Candida administration. Candida renal-I/R mice, when compared with non-Candida I/R, demonstrated more profound injuries, including (i) gut-leakage; FITC-dextran assay and serum (1→3)-ß-D-glucan (BG), (ii) systemic inflammation (serum cytokines), and (iii) neutrophil extracellular traps (NETs); gene expression of peptidyl arginase 4 (PAD4) and IL-1ß, nuclear morphology staining by 4',6-diamidino-2-phenylindole (DAPI) and co-staining of myeloperoxidase (MPO) with neutrophil elastase (NE) in peripheral blood neutrophils. Although renal excretory function (serum creatinine) and renal histology score were nondifferent between renal-I/R mice with and without Candida, prominent renal NETs (PAD4 and IL-1ß expression with MPO and NE co-staining) was demonstrated in Candida renal-I/R mice. Additionally, neutrophil activation by lipopolysaccharide (LPS) plus BG (LPS + BG), when compared with LPS alone, caused (i) NETs formation; dsDNA, DAPI-stained nuclear morphology and MPO with NE co-staining, (ii) inflammatory responses; Spleen tyrosine kinase (Syk) and NFκB expression, and (iii) reduced cell energy status (maximal respiratory capacity using extracellular flux analysis). Also, LPS + BG-activated NETs formation was inhibited by a dectin-1 inhibitor, supporting an impact of BG signaling. In conclusion, Candida-renal I/R demonstrated more prominent serum BG and LPS from gut translocation that increased systemic inflammation and NETs through TLR-4 and dectin-1 activation. The influence of gut fungi in AKI should be concerned.

Obesity Exacerbates Lupus Activity in Fc Gamma Receptor IIb Deficient Lupus Mice Partly through Saturated Fatty Acid-Induced Gut Barrier Defect and Systemic Inflammation.

The prevalence of obesity is increasing, and the coexistence of obesity and systemic lupus erythematosus (lupus) is possible. A high-fat diet (HFD) was orally administered for 6 months in female 8-week-old Fc gamma receptor IIb deficient (FcgRIIb-/-) lupus or age and gender-matched wild-type (WT) mice. Lupus nephritis (anti-dsDNA, proteinuria, and increased creatinine), gut barrier defect (fluorescein isothiocyanate dextran), serum lipopolysaccharide (LPS), serum interleukin (IL)-6, liver injury (alanine transaminase), organ fibrosis (liver and kidney pathology), spleen apoptosis (activated caspase 3), and aorta thickness (but not weight gain and lipid profiles) were more prominent in HFD-administered FcgRIIb-/- mice than the obese WT, without injury in regular diet-administered mice (both FcgRIIb-/- and WT). In parallel, combined palmitic acid (PA; a saturated fatty acid) with LPS (PA + LPS) induced higher tumor necrotic factor-α, IL-6, and IL-10 in the supernatant, inflammatory genes (inducible nitric oxide synthase and IL-1ß), reactive oxygen species (dihydroethidium), and glycolysis with reduced mitochondrial activity (extracellular flux analysis) when compared with the activation by each molecule alone in both FcgRIIb-/- and WT macrophages. However, the alterations of these parameters were more prominent in PA + LPS-administered FcgRIIb-/- than in the WT cells. In conclusion, obesity accelerated inflammation in FcgRIIb-/- mice, partly due to the more potent responses from the loss of inhibitory FcgRIIb against PA + LPS with obesity-induced gut barrier defect.

A Comparison Between 1 Day versus 7 Days of Sepsis in Mice with the Experiments on LPS-Activated Macrophages Support the Use of Intravenous Immunoglobulin for Sepsis Attenuation.

BACKGROUND: Because survival and death after sepsis are partly due to a proper immune adaptation and immune dysregulation, respectively, survivors and moribund mice after cecal ligation and puncture (CLP) sepsis surgery and in vitro macrophage experiments were explored. METHODS: Characteristics of mice at 1-day and 7-days post-CLP, the representative of moribund mice (an innate immune hyper-responsiveness) and survivors (a successful control on innate immunity), respectively. In parallel, soluble heat aggregated immunoglobulin (sHA-Ig), a representative of immune complex, was tested in lipopolysaccharide (LPS)-activated macrophages together with a test of intravenous immunoglobulin (IVIG), a molecule of adaptive immunity, on CLP sepsis mice. RESULTS: Except for a slight increase in alanine transaminase (liver injury), IL-10, endotoxemia, and gut leakage (FITC-dextran assay), most of the parameters in survivors (7-days post-CLP) were normalized, with enhanced adaptive immunity, including serum immunoglobulin (using serum protein electrophoresis) and activated immune cells in spleens (flow cytometry analysis). The addition of sHA-Ig in LPS-activated macrophages reduced supernatant cytokines, cell energy (extracellular flux analysis), reactive oxygen species (ROS), several cell activities (proteomic analysis), and Fc gamma receptors (FcgRs) expression. The loss of anti-inflammatory effect of sHA-Ig in LPS-activated macrophages from mice with a deficiency on Fc gamma receptor IIb (FcgRIIb-/-), the only inhibitory signaling of FcgRs family, when compared with wild-type macrophages, implying the FcgRIIb-dependent mechanism. Moreover, IVIG attenuated sepsis severity in CLP mice as evaluated by serum creatinine, liver enzyme (alanine transaminase), serum cytokines, spleen apoptosis, and abundance of dendritic cells in the spleen (24-h post-CLP) and survival analysis. CONCLUSION: Immunoglobulin attenuated LPS-activated macrophages, partly, through the reduced cell energy of macrophages and might play a role in sepsis immune hyper-responsiveness. Despite the debate over IVIG's use in sepsis, IVIG might be beneficial in sepsis with certain conditions.

BAM15, a Mitochondrial Uncoupling Agent, Attenuates Inflammation in the LPS Injection Mouse Model: An Adjunctive Anti-Inflammation on Macrophages and Hepatocytes.

Controlof immune responses through the immunometabolism interference is interesting for sepsis treatment. Then, expression of immunometabolism-associated genes and BAM15, a mitochondrial uncoupling agent, was explored in a proinflammatory model using lipopolysaccharide (LPS) injection. Accordingly, the decreased expression of mitochondrial uncoupling proteins was demonstrated by transcriptomic analysis on metabolism-associated genes in macrophages (RAW246.7) and by polymerase chain reaction in LPS-stimulated RAW246.7 and hepatocytes (Hepa 1-6). Pretreatment with BAM15 at 24 h prior to LPS in macrophages attenuated supernatant inflammatory cytokines (IL-6, TNF-α, and IL-10), downregulated genes of proinflammatory M1 polarization (iNOS and IL-1ß), upregulated anti-inflammatory M2 polarization (Arg1 and FIZZ), and decreased cell energy status (extracellular flux analysis and ATP production). Likewise, BAM15 decreased expression of proinflammatory genes (IL-6, TNF-α, IL-10, and iNOS) and reduced cell energy in hepatocytes. In LPS-administered mice, BAM15 attenuated serum cytokines, organ injury (liver enzymes and serum creatinine), and tissue cytokines (livers and kidneys), in part, through the enhanced phosphorylated αAMPK, a sensor of ATP depletion with anti-inflammatory property, in the liver, and reduced inflammatory monocytes/macrophages (Ly6C +ve, CD11b +ve) in the liver as detected by Western blot and flow cytometry, respectively. In conclusion, a proof of concept for inflammation attenuation of BAM15 through metabolic interference-induced anti-inflammation on macrophages and hepatocytes was demonstrated as a new strategy of anti-inflammation in sepsis.

Candida Administration Worsens Uremia-Induced Gut Leakage in Bilateral Nephrectomy Mice, an Impact of Gut Fungi and Organismal Molecules in Uremia.

The impact of gut fungi and (1→3)-ß-d-glucan (BG), a major fungal cell wall component, on uremia was explored by Candida albicans oral administration in bilateral nephrectomy (BiNx) mice because of the prominence of C. albicans in the human intestine but not in mice. As such, BiNx with Candida administration (BiNx-Candida) enhanced intestinal injury (colon cytokines and apoptosis), gut leakage (fluorescein isothiocyanate [FITC]-dextran assay, endotoxemia, serum BG, and bacteremia), systemic inflammation, and liver injury at 48 h postsurgery compared with non-Candida BiNx mice. Interestingly, uremia-induced enterocyte apoptosis was severe enough for gut translocation of viable bacteria, as indicated by culture positivity for bacteria in blood, mesenteric lymph nodes (MLNs), and other organs, which was more severe in BiNx-Candida than in non-Candida BiNx mice. Candida induced alterations in the gut microbiota of BiNx mice as indicated by (i) the higher fungal burdens in the feces of BiNx-Candida mice than in sham-Candida mice by culture methods and (ii) increased Bacteroides with decreased Firmicutes and reduced bacterial diversity in the feces of BiNx-Candida mice compared with non-Candida BiNx mice by fecal microbiome analysis. In addition, lipopolysaccharide plus BG (LPS+BG), compared with each molecule alone, induced high supernatant cytokine levels, which were enhanced by uremic mouse serum in both hepatocytes (HepG2 cells) and macrophages (RAW264.7 cells). Moreover, LPS+BG, but not each molecule alone, reduced the glycolysis capacity and mitochondrial function in HepG2 cells as determined by extracellular flux analysis. Additionally, a probiotic, Lactobacillus rhamnosus L34 (L34), attenuated disease severity only in BiNx-Candida mice but not in non-Candida BiNx mice, as indicated by liver injury and serum cytokines through the attenuation of gut leakage, the fecal abundance of fungi, and fecal bacterial diversity but not fecal Gram-negative bacteria. In conclusion, Candida enhanced BiNx severity through the worsening of gut leakage and microbiota alterations that resulted in bacteremia, endotoxemia, and glucanemia.IMPORTANCE The impact of fungi in the intestine on acute uremia was demonstrated by the oral administration of Candida albicans in mice with the removal of both kidneys. Because fungi in the mouse intestine are less abundant than in humans, a Candida-administered mouse model has more resemblance to patient conditions. Accordingly, acute uremia, without Candida, induced intestinal mucosal injury, which resulted in the translocation of endotoxin, a major molecule of gut bacteria, from the intestine into blood circulation. In acute uremia with Candida, intestinal injury was more severe due to fungi and the alteration in intestinal bacteria (increased Bacteroides with decreased Firmicutes), leading to the gut translocation of both endotoxin from gut bacteria and (1→3)-ß-d-glucan from Candida, which synergistically enhanced systemic inflammation in acute uremia. Both pathogen-associated molecules were delivered to the liver and induced hepatocyte inflammatory responses with a reduced energy production capacity, resulting in acute uremia-induced liver injury. In addition, Lactobacillus rhamnosus attenuated intestinal injury through reduced gut Candida and improved intestinal bacterial conditions.

Over-expression of miR-223 induces M2 macrophage through glycolysis alteration and attenuates LPS-induced sepsis mouse model, the cell-based therapy in sepsis.

The attenuation of hyper-inflammation in sepsis with the administration of anti-inflammatory macrophages is an interesting adjuvant therapy for sepsis. Because the induction of anti-inflammatory macrophages by microRNA (miR), a regulator of mRNA, has been mentioned, the exploration on miR-induced anti-inflammatory macrophages was performed. The over-expression of miR-223 and miR-146a in RAW264.7 induced M2 macrophage-polarization (anti-inflammatory macrophages) as evaluated by the enhanced expression of Arginase-1 and Fizz. However, miR-223 over-expressed cells demonstrated the more potent anti-inflammatory property against LPS stimulation as lesser iNOS expression, lower supernatant IL-6 and higher supernatant IL-10 compared with miR-146a over-expressed cells. Interestingly, LPS stimulation in miR-223 over-expressed cells, compared with LPS-stimulated control cells, demonstrated lower activity of glycolysis pathway and higher mitochondrial respiration, as evaluated by the extracellular flux analysis, and also down-regulated HIF-1α, an important enzyme of glycolysis pathway. In addition, the administration of miR-223 over-expressed macrophages with IL-4 pre-conditioning, but not IL-4 stimulated control cells, attenuated sepsis severity in LPS injected mice as evaluated by serum creatinine, liver enzymes, lung histology and serum cytokines. In conclusion, miR-223 interfered with the glycolysis pathway through the down-regulation of HIF-1α, resulting in the anti-inflammatory status. The over-expression of miR-223 in macrophages prevented the conversion into M1 macrophage polarization after LPS stimulation. The administration of miR-223 over-expressed macrophages, with IL-4 preconditioning, attenuated sepsis severity in LPS model. Hence, a proof of concept in the induction of anti-inflammatory macrophages through the cell-energy interference for sepsis treatment was proposed as a basis of cell-based therapy in sepsis.

Corrigendum: Candida Administration Worsens Cecal Ligation and Puncture-Induced Sepsis in Obese Mice Through Gut Dysbiosis Enhanced Systemic Inflammation, Impact of Pathogen-Associated Molecules From Gut Translocation and Saturated Fatty Acid.

[This corrects the article DOI: 10.3389/fimmu.2020.561652.].

Candida Administration Worsens Cecal Ligation and Puncture-Induced Sepsis in Obese Mice Through Gut Dysbiosis Enhanced Systemic Inflammation, Impact of Pathogen-Associated Molecules From Gut Translocation and Saturated Fatty Acid.

Obesity induces gut leakage and elevates serum lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria, through gut translocation. Because Candida albicans is prominent in human gut but not in mouse, C. albicans, a source of (1→3)-ß-D-glucan (BG) in gut contents, was administered in high-fat diet (HFD)-induced obese mice at 1 week before sepsis induction by cecal ligation and puncture (CLP). As such, sepsis in Candida-administered obese mice was more severe than obese mice without Candida as determined by mortality, organ injury (liver and kidney), serum cytokines, gut leakage, endotoxemia, serum BG, and fecal Gram-negative bacteria (microbiome analysis). Mice subjected to CLP and fed a HFD, but not treated with Candida demonstrated a similar mortality to non-obese mice with more severe gut leakage and higher serum cytokines. In vitro experiments demonstrated that LPS plus BG (LPS + BG) induced higher supernatant cytokines from hepatocytes (HepG2) and macrophages (RAW264.7), compared with the activation by each molecule alone, and were amplified by palmitic acid, a representative saturated fatty acid. The energy production capacity of HepG2 cells was also decreased by LPS + BG compared with LPS alone as evaluated by extracellular flux analysis. However, Lactobacillus rhamnosus L34 (L34) improved sepsis, regardless of Candida administration, through the attenuation of gut leakage and gut dysbiosis. In conclusion, an impact of gut Candida was demonstrated by Candida pretreatment in obese mice that worsened sepsis through (1) gut dysbiosis-induced gut leakage and (2) amplified systemic inflammation due to LPS, BG, and saturated fatty acid.