Lipocalin-2 (Lcn-2) Attenuates Polymicrobial Sepsis with LPS Preconditioning (LPS Tolerance) in FcGRIIb Deficient Lupus Mice

Abstract

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.

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

“…In addition, a neutral soluble glucan, a competitive Dectin-1 binding agent (at 150 μg/ml; InvivoGen), was incubated simultaneously with BG as a Dectin-1 inhibitor to explore the impact of Dectin-1, a BG receptor, on hepatocytes. Moreover, energy metabolism profiles of hepatocytes activated by PA simultaneously with LPS or with LPS + BG with glycolysis estimation through extracellular acidification rate and mitochondrial oxidative phosphorylation by oxygen consumption rate were performed using Seahorse XFp Analyzers (Agilent, Santa Clara, CA, United States) upon HepG2 at 1 × 10 4 cells/well ( 47 , 48 ).…”

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 qPCR was performed following previous publications [ 29 ]. Briefly, total RNA was prepared from the cell culture by Trizol (Thermo fisher Scientific) and quantified by Nano drop ND-1000 (Thermo fisher scientific).…”

“…In addition, ATP content in 2x10 4 cells/ well was determined by the Luminescent ATP Detection Assay (Abcam, Cambridge, UK) according to the manufacturer’s protocol [ 39 ]. In addition, energy metabolism profiles with estimation of glycolysis and mitochondrial oxidative phosphorylation with extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were performed, respectively, by Seahorse XFp Analyzers (Agilent, Santa Clara, CA, USA) upon the miR-transfected RAW264.7 at 1x10 4 cells/ well by Seahorse Wave 2.6 software as previously described [ 29 ]. Glycolysis and mitochondrial parameters was calculated using the generator program report of XF Glycolysis stress test and XF Cell mito stress test, respectively, based on these following equations; Glycolysis = ECAR between glucose and oligomycin–ECAR before glucose administration, glycolysis capacity = ECAR between oligomycin and 2-Deoxy-d-glucose (2-DG)–ECAR before glucose administration, glycolysis reserve = ECAR between oligomycin and 2-DG—ECAR between glucose and oligomycin, basal respiration = OCR before oligomycin–OCR after antimycin A/ rotinone, respiratory capacity (maximal respiration) = OCR between Carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) and antimycin A/ rotinone–OCR after antimycin A/ rotinone and respiratory reserve = OCR between FCCP and antimycin A/ rotinone–OCR before oligomycin.…”

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

“…There is an age-dependency in the development of lupus characteristics in FcgRIIb-/- mice, as anti-dsDNA, a major lupus auto-antibody, spontaneously develops in these mice as early as 16–24 wks old [ 5 , 6 , 7 ]. FcgRIIb-/- mice younger than 24 wks old are asymptomatic because of the undetectable anti-dsDNA [ 8 , 9 ]. The loss of inhibitory signaling in FcgRIIb-/- mice not only causes lupus, but also results in the hyperresponsiveness against pathogen molecules, including lipopolysaccharide (LPS).…”

“…The loss of inhibitory signaling in FcgRIIb-/- mice not only causes lupus, but also results in the hyperresponsiveness against pathogen molecules, including lipopolysaccharide (LPS). This is possibly due to the crosstalk of FcgRs (a receptor for Fc portion of immunoglobulin) and TLR-4 (a LPS receptor) [ 5 , 8 , 9 , 10 , 11 ].…”