Akihito Inatsu

Novel mechanism of C-reactive protein for enhancing mouse liver innate immunity†

Although C‐reactive protein (CRP) is a representative acute‐phase protein produced by hepatocytes, the role of CRP in liver innate immunity remains unclear. Using C57BL/6 mice, the present study investigated how CRP affects the functions of liver macrophages, Kupffer cells, and natural killer / natural killer T (NK/NKT) cells under various conditions, including Escherichia coli infection, septic shock, and multiorgan dysfunction induced by interleukin (IL)‐12/lipopolysaccharide (LPS) (generalized Shwartzman reaction [GSR]), and LPS‐induced lethal hepatitis in Propionibacterium acnes–primed mice. When mice were challenged with a lethal dose of E. coli, synthetic CRP peptide decreased the mortality without decreasing serum tumor necrosis factor (TNF), presumably by enhancing the phagocytic activity of Kupffer cells. Synthetic CRP greatly decreased the production of TNF and reactive oxygen species from Kupffer cells and thereby rescued mice after lethal LPS challenge. CRP also decreased the mortality from GSR and lethal hepatitis by inhibiting TNF production from Kupffer cells, especially phagocytosing Kupffer cells. However, interferon‐gamma production from NK/NKT cells was generally not so affected. CRP reportedly binds to FcγRI and FcγRII, and the injection of anti‐FcγRII/III Ab into mice abrogated TNF production from, but increased the phagocytic activity of, Kupffer cells. Furthermore, CRP pretreatment restored the decreased phagocytic activity of Kupffer cells in burn‐injured mice and decreased TNF production by Kupffer cells and thereby inhibited mortality after sublethal E. coli infection. If CRP was injected into mice at 1 hour after lethal E. coli challenge, it slightly but significantly increased the survival rate. Conclusion: CRP thus enhances the phagocytosis of Kupffer cells but decreases their TNF production in a complex manner in which the pathway by way of FcγRII may be involved. (HEPATOLOGY 2009.)

Functional alterations of liver innate immunity of mice with aging in response to CpG‐oligodeoxynucleotide

Immune functions of liver natural killer T (NKT) cells induced by the synthetic ligand α‐galactosylceramide enhanced age‐dependently; hepatic injury and multiorgan dysfunction syndrome (MODS) induced by ligand‐activated NKT cells were also enhanced. This study investigated how aging affects liver innate immunity after common bacteria DNA stimulation. Young (6 weeks) and old (50‐60 weeks) C57BL/6 mice were injected with CpG oligodeoxynucleotides (CpG‐ODN), and the functions of liver leukocytes were assessed. A CpG‐ODN injection into the old mice remarkably increased tumor necrosis factor (TNF) production in Kupffer cells, and MODS and lethal shock were induced, both of which are rarely seen in young mice. Old Kupffer cells showed increased Toll‐like receptor‐9 expression, and CpG‐ODN challenge augmented TNF receptor and Fas‐L expression in liver NKT cells. Experiments using mice depleted of natural killer (NK) cells by anti‐asialoGM1 antibody (Ab), perforin knockout mice, and mice pretreated with neutralizing interferon (IFN)‐γ Ab demonstrated the important role of liver NK cells in antitumor immunity. The production capacities of old mice for IFN‐γ, IFN‐α, and perforin were much lower than those of young mice, and the CpG‐induced antitumor cytotoxicity of liver NK cells lessened. Lethal shock and MODS greatly decreased in old mice depleted/deficient in TNF, FasL, or NKT cells. However, depletion of NK cells also decreased serum TNF levels and FasL expression of NKT cells, which resulted in improved hepatic injury and survival, suggesting that NK cells are indirectly involved in MODS/lethal shock induced by NKT cells. Neutralization of TNF did not reduce the CpG‐induced antitumor effect in the liver. Conclusion: Hepatic injury and MODS mediated by NKT cells via the TNF and FasL‐mediated pathway after CpG injection increased, but the antitumor activity of liver NK cells decreased with aging. (HEPATOLOGY 2008.)

Enhancement of Neutrophil Function by Interleukin-18 Therapy Protects Burn-Injured Mice from Methicillin-Resistant Staphylococcus aureus

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) infection is a grave concern in burn-injured patients. We investigated the efficacy of interleukin-18 (IL-18) treatment in postburn MRSA infection. Alternate-day injections of IL-18 into burn-injured C57BL/6 mice significantly increased their survival after MRSA infection and after methicillin-sensitive S. aureus infection. Although IL-18 treatment of burn-injured mice augmented natural IgM production before MRSA infection and gamma interferon (IFN-γ) production after MRSA infection, neither IgM nor IFN-γ significantly contributed to the improvement in mouse survival. IL-18 treatment increased/restored the serum tumor necrosis factor (TNF), IL-17, IL-23, granulocyte colony-stimulating factor (G-CSF), and macrophage inflammatory protein (MIP-2) levels, as well as the neutrophil count, after MRSA infection of burn-injured mice; it also improved impaired neutrophil functions, phagocytic activity, production of reactive oxygen species, and MRSA-killing activity. However, IL-18 treatment was ineffective against MRSA infection in both burn- and sham-injured neutropenic mice. Enhancement of neutrophil functions by IL-18 was also observed in vitro. Furthermore, when neutrophils from IL-18-treated burn-injured mice were adoptively transferred into nontreated burn-injured mice 2 days after MRSA challenge, survival of the recipient mice increased. NOD-SCID mice that have functionally intact neutrophils and macrophages (but not T, B, or NK cells) were substantially resistant to MRSA infection. IL-18 treatment increased the survival of NOD-SCID mice after burn injury and MRSA infection. An adoptive transfer of neutrophils using NOD-SCID mice also showed a beneficial effect of IL-18-activated neutrophils, similar to that seen in C57BL/6 mice. Thus, although neutrophil functions were impaired in burn-injured mice, IL-18 therapy markedly activated neutrophil functions, thereby increasing survival from postburn MRSA infection.

IL‐15‐induced CD8+CD122+ T cells increase antibacterial and anti‐tumor immune responses: implications for immune function in aged mice

We previously proposed that mouse CD8+CD122+ T cells and human CD57+ T cells, which increase with age and exhibit potent IFN‐γ production, represent a double‐edged sword as they play critical roles in host defense and the lethal IL‐12/LPS‐induced generalized Shwartzman reaction (GSR). However, our proposal was based solely on comparisons of young and old mice. In this study, we attempted to increase CD8+CD122+ T cells in young mice with exogenous IL‐15 and confirm their countervailing functions in young mice. After young mice (6 weeks) were injected with IL‐15, they showed significant increases in CD8+CD122+ T cells in the liver and spleen. Liver CD8+CD122+ T cells from IL‐15‐pretreated mice had a potent capacity to produce IFN‐γ after IL‐12 injection or Escherichia coli infection. IL‐15‐pretreated mice showed increased survival to E. coli infections and enhanced anti‐tumor activities against liver metastatic EL4 cells, as well as an exacerbation of the GSR. Correspondingly, liver CD8+CD122+ T cells produced more perforin than CD8+CD122− T cells in EL4‐inoculated mice. Unexpectedly, comparable IL‐15 treatment did not induce further increases in CD8+CD122+ T cells in aged mice and did not enhance their defenses against bacterial infection or tumor growth. Interestingly, however, nontreated, aged mice (50 weeks) showed twofold higher IL‐15 levels (but not TNF or IFN‐γ) in liver homogenates compared with young mice. Our results further support that CD8+CD122+ T cells, which are increased physiologically or therapeutically by IL‐15, are involved in antibacterial immunity, anti‐tumor immunity, and the GSR.

Polythiol-Containing, Recombinant Mannosylated-Albumin Is a Superior CD68+/CD206+ Kupffer Cell–Targeted Nanoantioxidant for Treatment of Two Acute Hepatitis Models

Since reactive oxygen species (ROS) derived from Kupffer cells (KC), especially CD68+ KC, play a key role in the induction of hepatic oxidative stress and injuries, we developed a polythiolated- and mannosylated human serum albumin (SH-Man-HSA), which functions as a novel nanoantioxidant for delivering thiol to CD68+ KC. In vitro electron paramagnetic resonance coupled with pharmacokinetics and immunohistochemical studies showed that SH-Man-HSA possessed powerful radical-scavenging activity and rapidly and selectively delivered thiols to the liver via mannose receptor (CD206) on CD68+ cells. SH-Man-HSA significantly improved the survival rate of concanavalin-A (Con-A)–treated mice. Moreover, SH-Man-HSA exhibited excellent hepatoprotective functions, not by decreasing tumor necrosis factor or interferon-γ production that is closely associated with Con-A–induced hepatitis, but by suppressing ROS production. Interestingly, the protective effect of SH-Man-HSA was superior to N-acetyl cysteine (NAC). This could be attributed to the difference in the inhibition of hepatic oxidative stress between the two antioxidants depending on their potential for thiol delivery to the liver. Similar results were also observed for acetaminophen (APAP)-induced hepatopathy models. Flow cytometric data further confirmed that an increase in F4/80+/ROS+ cells was dramatically decreased by SH-Man-HSA. The administration of SH-Man-HSA at 4 hours following a Con-A or APAP injection also exhibited a profound hepatoprotective action against these hepatitis models, whereas this was not observed for NAC. It can be concluded therefore that SH-Man-HSA has great potential for use in a rescue therapy for hepatopathy as a nanoantioxidant because of its ability to efficiently and rapidly deliver thiols to CD68+/CD206+ KC.

Cardiac dysfunction induced by experimental myocardial infarction impairs the host defense response to bacterial infection in mice because of reduced phagocytosis of Kupffer cells

OBJECTIVE This study was undertaken to investigate the effects of cardiac dysfunction induced by experimental myocardial infarction on the host defense response to bacterial infection and the role of Kupffer cells in mediating this response. METHODS Myocardial infarction was induced in C57BL/6 mice by ligation of the left anterior descending coronary artery. Mice were challenged with Escherichia coli intravenously 1, 5, and 14 days after myocardial infarction or sham operation. Thereafter, the cytokine production and the function of their Kupffer cells were assessed. RESULTS Mice with myocardial infarction showed remarkable cardiac dysfunction and had a significantly lower survival than sham mice after bacterial challenge at 5 days after surgery; bacterial challenge at 1 or 14 days after surgery resulted in no difference in survival between myocardial infarction and sham mice. The phagocytic activity of Kupffer cells, assessed by fluorescein isothiocyanate microspheres, remarkably decreased in mice with myocardial infarction 5 days after surgery. Serum peaks of tumor necrosis factor and interferon-gamma after bacterial challenge were also suppressed in mice with myocardial infarction at 5 days. Production of these cytokines and immunoglobulin-M from liver mononuclear cells was also impaired in mice with myocardial infarction. Enhancement of the phagocytic activity of Kupffer cells by C-reactive protein significantly improved survival after infection in mice with myocardial infarction, although neither interleukin-18 nor immunoglobulin-M treatment improved survival. CONCLUSIONS Cardiac dysfunction induced by myocardial infarction renders mice susceptible to bacterial infection and increases mortality because of a reduced ability of Kupffer cells to clear infectious bacteria. C-reactive protein-enhanced phagocytic activity of Kupffer cells may improve the poor prognosis after bacterial infection in mice with myocardial infarction.

The relationships between intradialytic hypotension and estimated peak oxygen uptake or heart rate recovery after exercise

キーワード:透析関連低血圧,心肺運動負荷試験,最高酸素摂取量,心拍数減衰応答 〈要旨〉 本研究の目的は,透析関連低血圧(IDH)の指標としての推定最高酸素摂取量(peak VO2),心拍数減衰応答(HRR) の有用性を明らかにすることである.対象は運動負荷試験を実施した透析患者 216名とした.非 IDH群に比し IDH 群の peak VO2,HRRは有意に低値を示した(p<0.001).血清アルブミン,心機能,動脈硬化指標で調整後も peak VO2(OR 0.80,95%CI 0.70‒0.91,p=0.001),HRR(OR 0.91,95%CI 0.85‒0.98,p=0.009)は IDHと関連を示し, カットオフ値は peak VO2 13.8 mL/kg/min(感度 0.86,特異度 0.69,p<0.001),HRR 7拍(感度 0.80,特異度 0.62, p<0.001)であった.peak VO2,HRRは IDHの独立したリスク因子であり,カットオフ値における予測精度も高 く,指標としての有用性が示された.