Retraction: α-GalCer ameliorates listeriosis by accelerating infiltration of Gr-1+ cells into the liver.

Retraction: Emoto, M., Emoto, Y., Yoshizawa, I., Kita, E., Shimizu, T., Hurwitz, R., Brinkmann, V. and Kaufmann, S.H.E. (2010), α-GalCer ameliorates listeriosis by accelerating infiltration of Gr-1+ cells into the liver. Eur. J. Immunol., 40: 1328-1341. DOI: https://doi.org/10.1002/eji.200939594 The above article, published online on 16 February 2010 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the Chairman of the Executive Committee of the European Journal of Immunology and Wiley-VCH Verlag GmbH & Co. KGaA. The retraction has been agreed following an investigation carried out by Gunma University (http://www.gunma-u.ac.jp/wp-content/uploads/2017/10/chosakekka29.pdf). The investigation was unable to determine the validity of the images for which Professor Emoto, the article's corresponding author, was responsible. As a result, the journal has made the decision to retract the article.

A Simple, Reproducible, Inexpensive, Yet Old-Fashioned Method for Determining Phagocytic and Bactericidal Activities of Macrophages.

Macrophages (Mφ) play a pivotal role in the protection system by recognizing and eliminating invading pathogenic bacteria. Phagocytosis and the killing of invading bacteria are major effector functions of Mφ. Although the phagocytic and bactericidal activities of Mφ have been analyzed via several methods using a light microscope, a fluorescence microscope, or a fluorescence-activated cell sorter, expensive materials and equipment are usually required, and the methods are rather complicated. Moreover, it is impossible to determine both the phagocytic and bactericidal activities of Mφ simultaneously using these methods. In this review, we describe a simple, reproducible, inexpensive, yet old-fashioned method (antibiotic protection assay) for determining the phagocytic and bactericidal activities of Mφ.

Listeriolysin O, but not Murine E-cadherin, is Involved in Invasion of Listeria monocytogenes into Murine Liver Parenchymal Cells.

Human E-cadherin and listeriolysin O (LLO) are involved in invasion of Listeria monocytogenes into human liver parenchymal cells (LPC). Yet, it remains to be determined whether murine E-cadherin and LLO participate in invasion of L. monocytogenes into murine LPC. In the present study, involvement of murine E-cadherin and LLO in invasion of L. monocytogenes into murine LPC was investigated. Murine E-cadherin was expressed on murine LPC, but the expression became undetectable by insertion of transgene of Simian virus 40 large T antigen. Although invasion of L. monocytogenes into murine LPC was found regardless of murine E-cadherin expression, infection rate of L. monocytogenes being unable to secrete LLO was lower than that of L. monocytogenes being capable of secreting LLO. Our RESULTS verify that invasion of L. monocytogenes into murine LPC occurs independently of murine E-cadherin and indicate that LLO participates in invasion of L. monocytogenes into murine LPC.

Acquired resistance of Listeria monocytogenes in and escaped from liver parenchymal cells to gentamicin is caused by being coated with their plasma membrane.

After systemic infection, a majority of Listeria monocytogenes invade liver parenchymal cells (LPC), replicate therein and spread to neighboring cells, suggesting that 3 different types of L. monocytogenes exist in the liver: L. monocytogenes being unable to invade LPC, residing in LPC, and escaped from infected LPC. Although listeriolysin O (LLO) participates in escape of L. monocytogenes from macrophages and L. monocytogenes is susceptible to gentamicin (Gm), it remains elusive whether LLO participates in invasion/escape of L. monocytogenes into/from LPC, and whether L. monocytogenes in/escaped from LPC are susceptible to Gm. In the present study, we examined whether LLO is involved in invasion/escape of L. monocytogenes into/from LPC and whether L. monocytogenes in/escaped from LPC are susceptible to Gm. Invasion/escape of L. monocytogenes were found in LPC lines regardless of LLO expression, and L. monocytogenes in/escaped from LPC lines showed resistance to Gm. L. monocytogenes escaped from LPC lines were coated with their plasma membrane and the acquired resistance to Gm was abrogated by saponin. Our results indicate that invasion/escape of L. monocytogenes into/from LPC occur independently of LLO, and suggest that the acquired resistance of L. monocytogenes in/escaped from LPC to Gm is caused by being coated with their plasma membrane.

Alpha-galactosylceramide promotes killing of Listeria monocytogenes within the macrophage phagosome through invariant NKT-cell activation.

alpha-Galactosylceramide (alpha-GalCer) has been exploited for the treatment of microbial infections. Although amelioration of infection by alpha-GalCer involves invariant natural killer T (iNKT)-cell activation, it remains to be determined whether macrophages (Mphi) participate in the control of microbial pathogens. In the present study, we examined the participation of Mphi in immune intervention in infection by alpha-GalCer using a murine model of listeriosis. Phagocytic and bactericidal activities of peritoneal Mphi from C57BL/6 mice, but not iNKT cell-deficient mice, were enhanced after intraperitoneal injection of alpha-GalCer despite the absence of iNKT cells in the peritoneal cavity. High levels of gamma interferon (IFN-gamma) and nitric oxide (NO) were detected in the peritoneal cavities of mice treated with alpha-GalCer and in culture supernatants of peritoneal Mphi from mice treated with alpha-GalCer, respectively. Although enhanced bactericidal activity of peritoneal Mphi by alpha-GalCer was abrogated by endogenous IFN-gamma neutralization, this was only marginally affected by NO inhibition. Similar results were obtained by using a listeriolysin O-deficient strain of Listeria monocytogenes. Moreover, respiratory burst in Mphi was increased after alpha-GalCer treatment. Our results suggest that amelioration of listeriosis by alpha-GalCer is, in part, caused by enhanced killing of L. monocytogenes within phagosomes of Mphi activated by IFN-gamma from iNKT cells residing in an organ(s) other than the peritoneal cavity.

Alpha-GalCer ameliorates listeriosis by accelerating infiltration of Gr-1+ cells into the liver.

Alpha-galactosylceramide (alpha-GalCer) activates invariant (i)NKT cells, which in turn stimulate immunocompetent cells. Although activation of iNKT cells appears critical for regulation of immune responses, it remains elusive whether protection against intracellular bacteria can be induced by alpha-GalCer. Here, we show that alpha-GalCer treatment ameliorates murine listeriosis, and inhibits inflammation following Listeria monocytogenes infection. Liver infiltration of Gr-1+ cells and gamma/delta T cells was accelerated by alpha-GalCer treatment. Gr-1+ cell and gamma/delta T-cell depletion exacerbated listeriosis in alpha-GalCer-treated mice, and this effect was more pronounced after depletion of Gr-1+ cells than that of gamma/delta T cells. Although GM-CSF and IL-17 were secreted by NKT cells after alpha-GalCer treatment, liver infiltration of Gr-1+ cells was not prevented by neutralizing mAb. In parallel to the numerical increase of CD11b+Gr-1+ cells in the liver following alpha-GalCer treatment, CD11b-Gr-1+ cells were numerically reduced in the bone marrow. In addition, respiratory burst in Gr-1+ cells was enhanced by alpha-GalCer treatment. Our results indicate that alpha-GalCer-induced antibacterial immunity is caused, in part, by accelerated infiltration of Gr-1+ cells and to a lesser degree of gamma/delta T cells into the liver. We also suggest that the infiltration of Gr-1+ cells is caused by an accelerated supply from the bone marrow.

Dissociated expression of natural killer 1.1 and T-cell receptor by invariant natural killer T cells after interleukin-12 receptor and T-cell receptor signalling.

Invariant (i) natural killer T (NKT) cells become undetectable after stimulation with alpha-galactosylceramide (alpha-GalCer) or interleukin (IL)-12. Although down-modulation of surface T-cell receptor (TCR)/NKR-P1C (NK1.1) expression has been shown convincingly after stimulation with alpha-GalCer, it is unclear whether this also holds true for IL-12 stimulation. To determine whether failure to detect iNKT cells after IL-12 stimulation is caused by dissociation/internalization of TCR and/or NKR-P1C, or by block of de novo synthesis of these molecules, and to examine the role of IL-12 in the disappearance of iNKT cells after stimulation with alpha-GalCer, surface (s)/cytoplasmic (c) protein expression, as well as messenger RNA (mRNA) expression of TCR/NKR-P1C by iNKT cells after stimulation with alpha-GalCer or IL-12, and the influence of IL-12 neutralization on the down-modulation of sTCR/sNKR-P1C expression by iNKT cells after stimulation with alpha-GalCer were examined. The s/cTCR(+ )s/cNKR-P1C(+) iNKT cells became undetectable after in vivo administration of alpha-GalCer, which was partially prevented by IL-12 neutralization. Whereas s/cNKR-P1C(+) iNKT cells became undetectable after in vivo administration of IL-12, s/cTCR(+) iNKT cells were only marginally affected. mRNA expression of TCR/NKR-P1C remained unaffected by alpha-GalCer or IL-12 treatment, despite the down-modulation of cTCR and/or cNKR-P1C protein expression. By contrast, cTCR(+ )cNKR-P1C(+) sTCR(-) sNKR-P1C(-) iNKT cells and cNKR-P1C(+) sNKR-P1C(-) iNKT cells were detectable after in vitro stimulation with alpha-GalCer and IL-12, respectively. Our results indicate that TCR and NKR-P1C expression by iNKT cells is differentially regulated by signalling through TCR and IL-12R. They also suggest that IL-12 participates, in part, in the disappearance of iNKT cells after stimulation with alpha-GalCer by down-modulating not only sNKR-P1C, but also sTCR.

Intracellular bacterial infection and invariant NKT cells.

The invariant (i) natural killer (NK)T cells represent a unique subset of T lymphocytes which express the V alpha 14 chain of the T cell receptor (TCR), that recognizes glycolipid antigens presented by the nonpolymorphic major histocompatibility complex (MHC) class I-like antigen presentation molecule CD1d, and they participate in protection against some microbial pathogens. Although iNKT cells have originally been regarded as T cells co-expressing NKR-P1B/C (NK1.1: CD 161), they do not seem to consistently express this marker, since NK1.1 surface expression on iNKT cells undergoes dramatic changes following facultative intracellular bacterial infection, which is correlated with functional changes of this cell population. Accumulating evidence suggests that NK1.1 allows recognition of "missing-self", thus controlling activation/inhibition of NK1.1-expressing cells. Therefore, it is tempting to suggest that iNKT cells participate in the regulation of host immune responses during facultative intracellular bacterial infection by controlling NK1.1 surface expression. These findings shed light not only on the unique role of iNKT cells in microbial infection, but also provide evidence for new aspects of the NK1.1 as a regulatory molecule on these cells.

Role of interleukin-12 in determining differential kinetics of invariant natural killer T cells in response to differential burden of Listeria monocytogenes.

Invariant (i) natural killer (NK) T cells are unique T lymphocytes expressing NKR-P1B/C (NK1.1), which recognize glycolipids, notably alpha-galactosylceramide (alpha-GalCer) presented by CD1d. The characteristic phenotype of these iNKT cells undergoes dramatic changes following Listeria monocytogenes infection, and interleukin (IL)-12 is involved in these alterations. Here we show that liver iNKT cells in mice are differentially influenced by the load of infection. Liver alpha-GalCer/CD1d tetramer-reactive (alpha-GalCer/CD1d(+)) T cells expressing NK1.1 became undetectable by day 2 following L. monocytogenes infection and concomitantly cells lacking NK1.1 increased regardless of the severity of infection. Whereas alpha-GalCer/CD1d(+)NK1.1(+) T cells remained virtually undetectable on day 4 following low-dose infection, considerable numbers of these cells were detected in high-dose-infected mice. Whereas numbers of IL-12 producers in the liver on day 4 post infection were comparable in low- and high-dose-infected mice without in vitro restimulation with heat-killed Listeria, those were more prominent in low-dose-infected mice than in high-dose-infected mice after restimulation despite the fact that higher numbers of macrophages and granulocytes infiltrated the liver in high-dose-infected mice than in low-dose-infected mice. Our results indicate that NK1.1 surface expression on iNKT cells is differentially modulated by the burden of infection, and suggest that a high bacterial load probably causes loss of IL-12 production.

Reversible NK1.1 surface expression on invariant liver natural killer T cells during Listeria monocytogenes infection.

The invariant (i) natural killer (NK)T cells consistently express the Valpha14 chain of the T cell receptor (TCR) and recognize alpha-galactosylceramide (alpha-GalCer) presented by the nonpolymorphic presentation molecule CD1d. Despite their name, the iNKT cells represent a heterogeneous population, which can be divided on the basis of NK1.1 surface expression. Here we show that NK1.1 surface expression on liver iNKT cells in mice fluctuates during Listeria monocytogenes infection. At early stages of listeriosis, iNKT cells expressing NK1.1 were numerically reduced and those lacking NK1.1 were increased. At later time points, the NK1.1(-) iNKT cell population contracted, whereas NK1.1(+) iNKT cells reemerged. Alterations in NK1.1 surface expression on iNKT cells were paralleled by numerical changes of interleukin (IL)-12 producers in the liver and were completely prevented by endogenous IL-12 neutralization, whereas NK1.1 surface alterations on iNKT cells following alpha-GalCer stimulation were not prevented. Adoptive cell transfer experiments revealed that the liver NK1.1(-) iNKT cells from NK1.1(+) cell-depleted L. monocytogenes-infected mice accumulated in the liver of recipient recombination-activating gene-1-deficient mice where they acquired NK1.1 surface expression. Thus, we present first evidence that NK1.1 surface expression on liver iNKT cells is reversible during L. monocytogenes infection, and that different mechanisms underlie stimulation by TCR and IL-12.

Rapid development of a gamma interferon-secreting glycolipid/CD1d-specific Valpha14+ NK1.1- T-cell subset after bacterial infection.

The phenotypic and functional changes of glycolipid presented by CD1d(glycolipid/CD1d) specific Valpha14+ T cells in the liver of mice at early stages of bacterial infection were investigated. After Listeria monocytogenes infection or interleukin-12 (IL-12) treatment, alpha-galactosylceramide/CD1d tetramer-reactive (alpha-GalCer/CD1d+) T cells coexpressing natural killer (NK) 1.1 marker became undetectable and, concomitantly, cells lacking NK1.1 emerged in both euthymic and thymectomized animals. Depletion of the NK1.1+ subpopulation prevented the emergence of alpha-GalCer/CD1d+ NK1.1- T cells. Before infection, NK1.1+, rather than NK1.1-, alpha-GalCer/CD1d+ T cells coexpressing CD4 were responsible for IL-4 production, whereas gamma interferon (IFN-gamma) was produced by cells regardless of NK1.1 or CD4 expression. After infection, IL-4-secreting cells became undetectable among alpha-GalCer/CD1d+ T cells, but considerable numbers of IFN-gamma-secreting cells were found among NK1.1-, but not NK1.1+, cells lacking CD4. Thus, NK1.1 surface expression and functional activities of Valpha14+ T cells underwent dramatic changes at early stages of listeriosis, and these alterations progressed in a thymus-independent manner. In mutant mice lacking all alpha-GalCer/CD1d+ T cells listeriosis was ameliorated, suggesting that the subtle contribution of the NK1.1- T-cell subset to antibacterial protection is covered by more profound detrimental effects of the NK1.1+ T-cell subset.

Lack of macrophage migration inhibitory factor protects mice against concanavalin A-induced liver injury.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is involved in inflammatory and immune-mediated diseases but the role of MIF in liver injury has not yet been elucidated. METHODS: We investigated biochemically, histologically and immunologically the character of MIF in concanavalin A (Con A)-induced T-cell-mediated liver injury using MIF knockout (KO) mice and wild-type (WT) mice. RESULTS: MIF KO mice showed significantly decreased serum alanine aminotransferase values and suppressed histological change with massive necrosis of the hepatic parenchymal cells and infiltration of inflammatory cells compared with their WT counterparts. This protection was not mediated by either tumor necrosis factor-alpha or interferon-gamma, which are critical mediators of Con A-induced liver injury, as their serum concentrations were shown to be similar in MIF KO and WT mice. On the other hand, a flow cytometric analysis demonstrated that the number of activated hepatic leukocytes decreased more in the MIF KO mice than in the WT mice. CONCLUSIONS: A lack of MIF protected the mice from Con A-induced liver injury. Controlling the MIF activity may be a useful therapeutic strategy for treating such T-cell activation-associated liver diseases as autoimmune hepatitis and viral hepatitis.

Functionally active CD8alphabeta+ TCRgammadelta intestinal intraepithelial lymphocytes in athymic nu/nu mice.

Murine intestinal intraepithelial lymphocytes (IEL) encompass a high proportion of TCRgammadelta cells. A vast majority of these TCRgammadelta IEL express CD8alpha, but not CD8beta (CD8alphaalpha homodimer), and are considered to develop in intestinal epithelial layers independently of a functional thymus. Here we show that TCRgammadelta cells expressing both CD8alpha and CD8beta (CD8alphabeta heterodimer) appear in athymic nu/nu mice, although their appearance is random. The IEL comprising CD8alphabeta(+) TCRgammadelta cells expressed pronounced cytolytic and IFN-gamma-producing activities after TCRgammadelta ligation, which were markedly stronger than activities of IEL lacking CD8alphabeta(+) TCRgammadelta cells. Purified CD8alphabeta(+) TCRgammadelta cells expressed strong cytolytic activities and produced large quantities of IFN-gamma after TCR engagement. CD8alphabeta(+) TCRgammadelta cells were also identified among IEL from euthymic C57BL/6 mice, although their abundance varied among individual animals. However, cytolytic and IFN-gamma-producing activities in euthymic C57BL/6 mice were markedly lower than those in athymic nu/nu mice. Our findings suggest that CD8alphabeta(+) TCRgammadelta cells can develop in the intestine independently of a functional thymus/thymic epithelial cells and that they perform biological functions in situ.

Highly biased type 1 immune responses in mice deficient in LFA-1 in Listeria monocytogenes infection are caused by elevated IL-12 production by granulocytes.

LFA-1 (CD11a/CD18) plays a key role in various inflammatory responses. Here we show that the acquired immune response to Listeria monocytogenes is highly biased toward type 1 in the absence of LFA-1. At the early stage of listeriosis, numbers of IFN-gamma producers in the liver and spleen of LFA-1(-/-) mice were markedly increased compared with heterozygous littermates and Valpha14(+)NKT cell-deficient mice, and NK cells were major IFN-gamma producers. Numbers of IL-12 producers were also markedly elevated in LFA-1(-/-) mice compared with heterozygous littermates, and endogenous IL-12 neutralization impaired IFN-gamma production by NK cells. Granulocyte depletion diminished numbers of IL-12 producers and IFN-gamma-secreting NK cells in the liver of LFA-1(-/-) mice. Granulocytes from the liver of L. monocytogenes-infected LFA-1(-/-) mice were potent IL-12 producers. Thus, in the absence of LFA-1, granulocytes are a major source of IL-12 at the early stage of listeriosis. We assume that highly biased type 1 immune responses in LFA-1(-/-) mice are caused by increased levels of IL-12 from granulocytes and that granulocytes play a major role in IFN-gamma secretion by NK cells. In conclusion, LFA-1 regulates type 1 immune responses by controlling prompt infiltration of IL-12-producing granulocytes into sites of inflammation.

Increased resistance of LFA-1-deficient mice to lipopolysaccharide-induced shock/liver injury in the presence of TNF-alpha and IL-12 is mediated by IL-10: a novel role for LFA-1 in the regulation of the proinflammatory and anti-inflammatory cytokine balance.

Challenge with low doses of LPS together with D-galactosamine causes severe liver injury, resulting in lethal shock (low dose LPS-induced shock). We examined the role of LFA-1 in low dose LPS-induced shock. LFA-1(-/-) mice were more resistant to low dose LPS-induced shock/liver injury than their heterozygous littermates, although serum levels of TNF-alpha and IL-12 were higher in these mice. C57BL/6 mice were not rescued from lethal effects of LPS by depletion of NK1(+) cells, granulocytes, or macrophages, and susceptibility of NKT cell-deficient mice was comparable to that of controls. High numbers of platelets were detected in the liver of LFA-1(+/-) mice after low dose LPS challenge, whereas liver accumulation of platelets was only marginal in LFA-1(-/-) mice. Following low dose LPS challenge, serum levels of IL-10 were higher in LFA-1(-/-) mice than in LFA-1(+/-) mice, and susceptibility to low dose LPS-induced shock as well as platelet accumulation in the liver of LFA-1(-/-) mice were markedly increased by IL-10 neutralization. Serum levels of IL-10 in LFA-1(+/-) mice were only marginally affected by macrophage depletion. However, in LFA-1(-/-) mice macrophage depletion markedly reduced serum levels of IL-10, and as a corollary, susceptibility of LFA-1(-/-) mice to low dose LPS-induced shock was markedly elevated despite the fact that TNF-alpha levels were also diminished. We conclude that LFA-1 participates in LPS-induced lethal shock/liver injury by regulating IL-10 secretion from macrophages and that IL-10 plays a decisive role in resistance to shock/liver injury. Our data point to a novel role of LFA-1 in control of the proinflammatory/anti-inflammatory cytokine network.

Neutrophilia in LFA-1-deficient mice confers resistance to listeriosis: possible contribution of granulocyte-colony-stimulating factor and IL-17.

LFA-1 (CD11a/CD18) plays a crucial role in various inflammatory responses. In this study, we show that LFA-1(-/-) mice are far more resistant to Listeria monocytogenes infection than LFA-1(+/-) mice. Consistent with this, we found the following: 1) the numbers of granulocytes infiltrating the liver were markedly higher in LFA-1(-/-) mice than in LFA-1(+/-) mice, 2) increased antilisterial resistance in LFA-1(-/-) mice was abrogated by depletion of granulocytes, and 3) the numbers of granulocytes in peripheral blood, and the serum levels of both G-CSF and IL-17 were higher in LFA-1(-/-) mice than in LFA-1(+/-) mice. Neither spontaneous apoptosis nor survival of granulocytes from LFA-1(-/-) mice were affected by physiological concentrations of G-CSF. Our data suggest regulatory effects of LFA-1 on G-CSF and IL-17 secretion, and as a corollary on neutrophilia. Consequently, we conclude that increased resistance of LFA-1(-/-) mice to listeriosis is due to neutrophilia facilitating liver infiltration by granulocytes promptly after L. monocytogenes infection, although it is LFA-1 independent.

Critical role of NK cells rather than V alpha 14(+)NKT cells in lipopolysaccharide-induced lethal shock in mice.

Although macrophages play a central role in the pathogenesis of septic shock, NK1(+) cells have also been implicated. NK1(+) cells comprise two major populations, namely NK cells and V alpha 14(+)NKT cells. To assess the relative contributions of these NK1(+) cells to LPS-induced shock, we compared the susceptibility to LPS-induced shock of beta(2)-microglobulin (beta(2)m)(-/-) mice that are devoid of V alpha 14(+)NKT cells, but not NK cells, with that of wild-type (WT) mice. The results show that beta(2)m(-/-) mice were more susceptible to LPS-induced shock than WT mice. Serum levels of IFN-gamma following LPS challenge were significantly higher in beta(2)m(-/-) mice, and endogenous IFN-gamma neutralization or in vivo depletion of NK1(+) cells rescued beta(2)m(-/-) mice from lethal effects of LPS. Intracellular cytokine staining revealed that NK cells were major IFN-gamma producers. The J alpha 281(-/-) mice that are exclusively devoid of V alpha 14(+)NKT cells were slightly more susceptible to LPS-induced shock than heterozygous littermates. Hence, LPS-induced shock can be induced in the absence of V alpha 14(+)NKT cells and IFN-gamma from NK cells is involved in this mechanism. In WT mice, hierarchic contribution of different cell populations appears likely.