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The influence of gut-leakage or gut-microbiota upon lupus progression was explored in 2 lupus mouse models. Pristane, administered in 4-wk-old wild-type (WT) female mice, induced lupus characteristics at 24-wk-old similar to the lupus-onset in FcGRIIb−/− mice. Gut-microbiota alteration was induced by co-housing together with the gavage of feces from 40-wk-old FcGRIIb−/− mice (symptomatic lupus). On the other hand, gut-leakage was induced by dextran sulfate solution (DSS). DSS and gut-microbiota alteration induced high serum anti-dsDNA immunoglobulin (Ig) as early as 30 days post-DSS only in FcGRIIb−/− mice. DSS, but not gut-microbiota alteration, enhanced lupus characteristics (serum creatinine and proteinuria) in both lupus models (but not in WT) at 60 days post-DSS. Indeed, DSS induced the translocation of molecular components of gut-pathogens as determined by bacterial burdens in mesenteric lymph node (MLN), endotoxemia (gut-bacterial molecule) and serum (1→3)-β-D-glucan (BG) (gut-fungal molecule) as early as 15 days post-DSS together with enhanced MLN apoptosis in both WT and lupus mice. However, DSS induced spleen apoptosis in FcGRIIb−/− and WT mice at 30 and 60 days post-DSS, respectively, suggesting the higher impact of gut-leakage against spleen of lupus mice. In addition, macrophages preconditioning with LPS plus BG were susceptible to starvation-induced apoptosis, predominantly in FcGRIIb−/− cell, implying the influence of gut-leakage upon cell stress. In summary, gut-leakage induced gut-translocation of organismal-molecules then enhanced the susceptibility of stress-induced apoptosis, predominantly in lupus. Subsequently, the higher burdens of apoptosis in lupus mice increased anti-dsDNA Ig and worsen lupus severity through immune complex deposition. Hence, therapeutic strategies addressing gut-leakage in lupus are interesting.

Because both endotoxemia and gut dysbiosis post-splenectomy might be associated with systemic infection, the susceptibility against infection was tested by dextran sulfate solution (DSS)-induced colitis and lipopolysaccharide (LPS) injection models in splenectomy mice with macrophage experiments. Here, splenectomy induced a gut barrier defect (FITC-dextran assay, endotoxemia, bacteria in mesenteric lymph nodes, and the loss of enterocyte tight junction) and gut dysbiosis (increased Proteobacteria by fecal microbiome analysis) without systemic inflammation (serum IL-6). In parallel, DSS induced more severe mucositis in splenectomy mice than sham-DSS mice, as indicated by mortality, stool consistency, gut barrier defect, serum cytokines, and blood bacterial burdens. The presence of green fluorescent-producing (GFP) E. coli in the spleen of sham-DSS mice after an oral gavage supported a crucial role of the spleen in the control of bacteria from gut translocation. Additionally, LPS administration in splenectomy mice induced lower serum cytokines (TNF-α and IL-6) than LPS-administered sham mice, perhaps due to LPS tolerance from pre-existing post-splenectomy endotoxemia. In macrophages, LPS tolerance (sequential LPS stimulation) demonstrated lower cell activities than the single LPS stimulation, as indicated by the reduction in supernatant cytokines, pro-inflammatory genes (iNOS and IL-1β), cell energy status (extracellular flux analysis), and enzymes of the glycolysis pathway (proteomic analysis). In conclusion, a gut barrier defect after splenectomy was vulnerable to enterocyte injury (such as DSS), which caused severe bacteremia due to defects in microbial control (asplenia) and endotoxemia-induced LPS tolerance. Hence, gut dysbiosis and gut bacterial translocation in patients with a splenectomy might be associated with systemic infection, and gut-barrier monitoring or intestinal tight-junction strengthening may be useful.

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.

Inflammatory response plays an essential role in the resolution of infections. However, inflammation can be detrimental to an organism and cause irreparable damage. For example, during sepsis, a cytokine storm can lead to multiple organ failures and often results in death. One of the strongest triggers of the inflammatory response is bacterial lipopolysaccharides (LPS), acting mostly through Toll-like receptor 4 (TLR4). Paradoxically, while exposure to LPS triggers a robust inflammatory response, repeated or prolonged exposure to LPS can induce a state of endotoxin tolerance, a phenomenon where macrophages and monocytes do not respond to new endotoxin challenges, and it is often associated with secondary infections and negative outcomes. The cellular mechanisms regulating this phenomenon remain elusive. We used metabolic measurements to confirm differences in the cellular metabolism of na&iuml, ve macrophages and that of macrophages responding to LPS stimulation or those in the LPS-tolerant state. In parallel, we performed an unbiased secretome survey using quantitative mass spectrometry during the induction of LPS tolerance, creating the first comprehensive secretome profile of endotoxin-tolerant cells. The secretome changes confirmed that LPS-tolerant macrophages have significantly decreased cellular metabolism and that the proteins secreted by LPS-tolerant macrophages have a strong association with cell survival, protein metabolism, and the metabolism of reactive oxygen species.

Macrophages are responsible for the recognition of pathogen molecules. The downstream signalling of the innate immune responses against pathogen molecules, lipopolysaccharide (LPS) and (1→3)-β-D-glucan (BG), and the adaptive immune response to antibodies, Fc gamma receptor (FcgR), is spleen tyrosine kinase (Syk). Because pathogen molecules and antibodies could be presented in lupus, impact of Syk and macrophages in lupus is explored. FcgR-IIb deficient (FcgRIIb-/-) mice, a model of inhibitory signalling loss, at 40 weeks old, but not pristane mice (a chemical induction lupus model) demonstrated spontaneous elevation of LPS and BG in serum from gut translocation despite the similarity in faecal microbiome analysis. Syk abundance in FcgRIIb–/– mice was higher than in pristane mice, possibly due to several Syk activators (anti-dsDNA, LPS and BG), and Syk inhibitor–attenuated proteinuria and serum cytokines only in FcgRIIb–/– mice. In addition, LPS + BG enhanced the expression of activating FcgRs, NF-κB and Syk, together with supernatant TNF-α predominantly in FcgRIIb–/– compared to wild-type macrophages. The inhibitors against Dectin-1, Syk and nuclear factor kappa B, but not anti-Raf-1, reduced supernatant TNF-α in LPS+BG-activated macrophages, implying Syk-dependent signalling. The pathogen molecules enhanced activating-FcgRs, without inhibition, through Syk, a shared downstream innate and adaptive signalling, is responsible for the hyper-responsiveness in FcgRIIb–/– macrophages. In conclusion, Syk inhibitor attenuated inflammation in FcgRIIb–/– but not in pristane mice, implying the influence of a lupus genetic background in treatment modalities.

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)-β-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.

In patients with active lupus, spontaneous endotoxemia and possibly tolerance to lipopolysaccharide (LPS) is a potentially adverse complication. Similarly, previous reports have demonstrated that FcGRIIb deficient mice (FcGRIIb-/, a lupus mouse model) are susceptible to LPS tolerance-induced decreased cytokine responses that inadequate for the organismal control. Thus, understanding the relationship between FcGRIIb and LPS tolerance could improve the therapeutic strategy for lupus. LPS tolerance can be induced through sequential LPS stimulations in either cells or a model organism. In RAW264.7 (a mouse macrophage cell-line), sequential LPS stimulation induced the secretion of Lipocalin-2 (Lcn-2) despite reduced cytokine secretion and severe energy depletion, as measured by the extracellular flux analysis, typical of LPS tolerance. In contrast, treatment with recombinant Lcn-2 (rLcn-2) attenuated LPS tolerance, as shown by an increase in secreted cytokines and altered macrophage polarization toward M1 (increased iNOS and TNF-&alpha, ) in RAW264.7 cells. These results suggest a role of Lcn-2 in LPS tolerance attenuation. In bone marrow derived macrophages, Lcn-2 level was similar in LPS tolerant FcGRIIb-/- and wild-type (WT) cells despite the increased LPS tolerance of FcGRIIb-/- cells, suggesting relatively low basal levels of Lcn-2 produced in FcGRIIb-/- cells. In addition, attenuation of LPS tolerance effectuated by granulocyte-monocyte colony stimulating factor (GM-CSF) reduced Lcn-2 in both cell types, implying an inverse correlation between Lcn-2 and the severity of LPS tolerance. Consequently, rLcn-2 improved LPS tolerance only in FcGRIIb-/- macrophages and attenuated disease severity of cecal ligation and puncture (CLP) sepsis pre-conditioning with sequential LPS injection (LPS-CLP model) only in FcGRIIb-/- mice, but not in WT mice. To summarize, inadequate Lcn-2 production in FcGRIIb-/- macrophage might, at least in part, be responsible for the inordinate LPS tolerance compared with WT cells. Additionally, supplementation of rLcn-2 attenuates LPS tolerance in FcGRIIb-/- macrophages in vitro, and in FcGRIIb-/- mice with LPS-CLP sepsis in vivo. In conclusion, Lcn-2 secreted by macrophages is possibly an autocrine signal to counter the reduced cytokine secretion in LPS tolerance.

To investigate the role of Delta-like ligand 4 (DLL4) on tumour growth in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC)We suppressedEighteen days after implantation, tumour volume in mice implanted with shDLL4 HepG2.2.15 was significantly smaller than in mice implanted with control HepG2.2.15 (This study demonstrates that DLL4 is important in regulating the tumour growth of HBV-associated HCC as well as the neovascularization and suppression of HBV replication.

Background An early sepsis-induced acute kidney injury (sepsis-AKI) biomarker is currently in needed. Urinary neutrophil gelatinase-associated lipocalin (uNGAL) is a candidate of sepsis-AKI biomarker but with different cut-point values. Urinary exosomal activating transcriptional factor 3 (uATF3) has been mentioned as an interesting biomarker. Methods We conducted experiments in mice and a prospective, multicenter study in patients as a proof of concept that urine exosome is an interesting biomarker. An early expression of ATF3 in kidney of CD-1 mice at 6 h after cecal ligation and puncture implied the possibility of uATF3 as an early sepsis-AKI biomarker. Increase serum creatinine (Scr) ≥0.3 mg/dL from the baseline was used as an AKI diagnosis and urine was analyzed for uATF3 and uNGAL. Patients with baseline Scr at admission ≥1.5 mg/dL were excluded. Results The analysis showed higher Scr, uNGAL and uATF3 in patients with sepsis-AKI in comparison with patients with sepsis-non-AKI and healthy volunteers. A fair correlation, r2 = 0.47, between uATF3 and uNGAL was showed in sepsis-AKI group with Scr ≥2 mg/dL. To see if uATF3 could be an early sepsis-AKI biomarker, urine sample was collected daily during the first week of the admission. In sepsis-AKI and sepsis-non-AKI groups, uNGAL were 367 ± 43 ng/mL and 183 ± 23 ng/mL, respectively; and uATF3 were 19 ± 4 ng/mL and 1.4 ± 0.8 ng/mL, respectively. With the mean value of uNGAL and uATF3 in sepsis AKI as a cut-off level, AUROC of uNGAL and uATF3 were 64% (95% CI 0.54 to 0.74) and 84% (95% CI 0.77 to 0.91), respectively. Conclusions Urine exosome is an interesting source of urine biomarker and uATF3 is an interesting sepsis-AKI biomarker.

Serum neutrophil gelatinase associated lipocalin (sNGAL), a promising acute kidney injury (AKI) biomarker produced by renal and non-renal tissues, might be affected by sepsis. We evaluated sNGAL in zero glomerular filtration rate models [bilateral ureter obstruction (BUO) and bilateral nephrectomy (BiNx)] with subsequent cecal ligation and puncture (CLP)-induced sepsis in 6 to 8-week-old ICR mice. We found that sNGAL increased earlier than serum creatinine (Scr) in BiNx/BUO with and without CLP. The earliest time-point of increased sNGAL in BiNx+CLP was 1 h after surgery. Scr, but not sNGAL, was lower at 18 h after BiNx/BUO+CLP compared with BiNx/BUO alone. Compared with BUO, BiNx had higher, and equal sNGAL at 1 to 18 h and 36 h, respectively. Additionally, similar NGAL expression in internal organs (heart, lung, liver, and spleen) and survival rates indicated the comparable severity of BiNx and BUO. Serum interleukin (IL)-6 was increased and correlated with sNGAL in BiNx/BUO with and without sepsis. In summary, we demonstrated: sNGAL is an early AKI biomarker, which is not affected by sepsis; sNGAL is mainly produced by extrarenal sources as demonstrated by the comparable sNGAL in BiNx and BUO; the saturation of renal NGAL re-absorption in BUO is demonstrated by lower sNGAL in BUO at 1 to 18 h, but not at 36 h when compared with BiNx; and a correlation of sNGAL and IL-6 implied sNGAL is a good sepsis prognostic biomarker. Therefore, sNGAL is a more beneficial sepsis-AKI biomarker than Scr.