Hyper-elevated immune response of FcGRIIb-/- mice, a lupus model with an inhibitory-signaling defect, can become exhausted (less subsequent immune-response than the first response) with sequential lipopolysaccharide (LPS) stimulation. Endotoxin tolerance-related modifications of inflammatory response were investigated in FcGRIIb-/- mice in both an in vivo sepsis model and in vitro using cultured macrophages. Serum cytokine concentrations, after the second LPS injection (at 5-fold higher levels than the first dose), did not exceed the first dose levels in either FcGRIIb-/- or wild-type mice. These data indicated an endotoxin-tolerance response in both genetic backgrounds. However, the difference of cytokine levels between the first and second LPS injection was more prominent in FcGRIIb-/- mice. More importantly, CLP-induced sepsis after LPS-preconditioning (two separated doses of LPS administration) was more severe in FcGRIIb-/- mice (as measured by mortality rate, bacteria count in blood, serum cytokines, creatinine, and alanine transaminase). An attenuated response was demonstrated after two sequential LPS stimulations of bone-marrow-derived macrophages. Cytokine production was reduced and lower bacterial killing activity occurred with macrophages from FcGRIIb-/- mice relative to wild-type macrophages. Thus, there is a more prominent effect of endotoxin-tolerance in FcGRIIb-/- macrophages relative to wild-type. In conclusion, repeated-LPS administrations induced quantitatively greater endotoxin-tolerance responses in FcGRIIb-/- mice both in vivo and in vitro. Endotoxin-tolerance in vivo was associated with more severe sepsis, at least in part, due to macrophage-dysfunction.

BackgroundGold nanoparticles (AuNP) have several biochemical advantageous properties especially for a candidate of drug carrier. However, the non-conjugated AuNP has a higher rate of cellular uptake than the conjugated ones. Spherical AuNP in a proper size (20–30 nm) is non-toxic to mice and shows anti-inflammatory properties. We tested if the administration of AuNP, as an adjuvant to antibiotics, could attenuate bacterial sepsis in cecal ligation and puncture (CLP) mouse model with antibiotic (imipenem/cilastatin).ResultsIndeed, AuNP administration at the time of CLP improved the survival, blood bacterial burdens, kidney function, liver injury and inflammatory cytokines (TNF-α, IL-6, IL-1β and IL-10). AuNP also decreased M1 macrophages (CD86 + ve in F4/80 + ve cells) and increased M2 macrophages (CD206 + ve in F4/80 + ve cells) in the spleens of sepsis mice. The weak antibiotic effect of AuNP was demonstrated as the reduction of E. coli colony after 4 h incubation. In addition, AuNP altered cytokine production of bone-marrow-derived macrophages including reduced TNF-α, IL-6 and IL-1β but increased IL-10 at 6 and 24 h. Moreover, AuNP induced macrophage polarization toward anti-inflammatory responses (M2) as presented by increased Arg1 (Arginase 1) and PPARγ with decreased Nos2 (inducible nitric oxide synthase, iNos) and Nur77 at 3 h after incubation in vitro.ConclusionsThe adjuvant therapy of AuNP, with a proper antibiotic, attenuated CLP-induced bacterial sepsis in mice, at least in part, through the antibiotic effect and the induction of macrophage function toward the anti-inflammatory responses.

Obesity, a major healthcare problem worldwide, induces metabolic endotoxemia through the gut translocation of lipopolysaccharides (LPS), a major cell wall component of Gram-negative bacteria, causing a chronic inflammatory state. A combination of several probiotics including Lactobacillus acidophilus 5 (LA5), a potent lactic acid-producing bacterium, has previously been shown to attenuate obesity. However, data on the correlation between a single administration of LA5 versus microbiota alteration might be helpful for the probiotic adjustment. LA5 was administered daily together with a high-fat diet (HFD) for 8 weeks in mice. Furthermore, the condition media of LA5 was also tested in a hepatocyte cell-line (HepG2 cells). Accordingly, LA5 attenuated obesity in mice as demonstrated by weight reduction, regional fat accumulation, lipidemia, liver injury (liver weight, lipid compositions, and liver enzyme), gut permeability defect, endotoxemia, and serum cytokines. Unsurprisingly, LA5 improved these parameters and acidified fecal pH leads to the attenuation of fecal dysbiosis. The fecal microbiome analysis in obese mice with or without LA5 indicated; (i) decreased Bacteroidetes (Gram-negative anaerobes that predominate in non-healthy conditions), (ii) reduced total fecal Gram-negative bacterial burdens (the sources of gut LPS), (iii) enhanced Firmicutes (Gram-positive bacteria with potential benefits) and (iv) increased Verrucomycobia, especially Akkermansia muciniphila, a bacterium with the anti-obesity property. With LA5 administration, A. muciniphila in the colon were more than 2,000 folds higher than the regular diet mice as determined by 16S rRNA. Besides, LA5 produced anti-inflammatory molecules with a similar molecular weight to LPS that reduced cytokine production in LPS-activated HepG2 cells. In conclusion, LA5 attenuated obesity through (i) gut dysbiosis attenuation, partly through the promotion of A. muciniphila (probiotics with the difficulty in preparation processes), (ii) reduced endotoxemia, and (iii) possibly decreased liver injury by producing the anti-inflammatory molecules.

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-α) 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.

Dysfunction of FcGRIIb, the only inhibitory receptor of the FcGR family, is commonly found in the Asian population and is possibly responsible for the extreme endotoxin exhaustion in lupus. Here, the mechanisms of prominent endotoxin (LPS) tolerance in FcGRIIb−/− mice were explored on bone marrow-derived macrophages using phosphoproteomic analysis. As such, LPS tolerance decreased several phosphoproteins in the FcGRIIb−/− macrophage, including protein kinase C-β type II (PRKCB), which was associated with phagocytosis function. Overexpression of PRKCB attenuated LPS tolerance in RAW264.7 cells, supporting the role of this gene in LPS tolerance. In parallel, LPS tolerance in macrophages and in mice was attenuated by phorbol 12-myristate 13-acetate (PMA) administration. This treatment induced several protein kinase C families, including PRKCB. However, PMA attenuated the severity of mice with cecal ligation and puncture on LPS tolerance preconditioning in FcGRIIb−/− but not in wild-type cells. The significant reduction of PRKCB in the FcGRIIb−/− macrophage over wild-type cell possibly induced the more severe LPS-exhaustion and increased the infection susceptibility in FcGRIIb−/− mice. PMA induced PRKCB, improved LPS-tolerance, and attenuated sepsis severity, predominantly in FcGRIIb−/− mice. PRKCB enhancement might be a promising strategy to improve macrophage functions in lupus patients with LPS-tolerance from chronic infection.