key: cord-0009013-zivj0rm8
authors: Lucchiari, Maria A.; Pereira, Carlos A.
title: A Major Role of Macrophage Activation by Interferon-Gamma During Mouse Hepatitis Virus Type 3 Infection: II. Age-Dependent Resistance
date: 2011-11-02
journal: Immunobiology
DOI: 10.1016/s0171-2985(11)80163-4
sha: 1043007c52a94b83cc808e655d3d0745a0eee13b
doc_id: 9013
cord_uid: zivj0rm8

In contrast to adult mice, young AJ/mice, developed an acute hepatitis following infection with Mouse Hepatitis virus type 3. 100 % of the young animals died 4 to 5 days after the infection and high levels of virus were found in the liver and peritoneal exudate. Very low levels of IFN-$#x03B3; were found in the serum and peritoneal exudate of infected young mice. This was in contrast to the levels observed in adult mice. Spleen cells and macrophage cultures from young A/J mice, again in contrast to adult A/J mice, were shown to be unable to synthesize IFN-$#x03B3; and IFN-α/β respectively. Macrophages from either young or adult A/J mice were able to be activated with exogenous recombinant IFN-$#x03B3; or IFN-α/β, enabling both sets of cells to restrict MHV3 replication. The results indicate that the ability of the immune system to synthesize IFN-$#x03B3; and IFN-α/β may playa major role in the age-dependent resistance of A/J mice to MHV3.

MHV3 constitutes a model of viral infection in which resistance depends on the age and the genetic background of the animal (1) (2) (3) (4) (5) . AI] mice have been reported to be susceptible up to 3 weeks of age, developing an acute hepatitis and dying 4 to 5 days after infection.

Complete resistance develops after the 3rd week of life. After MHV3 infection, adult AI] mice show a mild disease which disappears 4 to 6 days later. Three types of mature cells has been shown to be required for transfering MHV3 resistance into young AI] mice: T lymphocytes, adherent spleen cells and a third population that shares several features with This work was supported in part by grants from the Funda<;ao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) and Conselho Nacional de Pesquisas (CNPq). M.A.L. was a recipient of a Deutscher Akademischer Austauschdienst (DAAD) fellowship during part of this work.

Abbreviations: MHV3=mouse hepatitis virus type 3; IFN=interferon; LPS=lipopolysaccharide; Con A = concanavalin A; FCS=fetal calf serum; PFU=plaque forming units; ip = intraperitoneally. natural killer cells (3, 5) . T lymphocytes and adherent cells such as peritoneal macrophages and Kupffer cells have been suggested by several authors to participate in the resistance against MHV3 (2, 4, 6-10), but natural killer cells have not been thought to be of overWhelming importance in the defense of mice against MHV3 (11, 12) .

Depending on the genetic background of the adult animal, different patterns of susceptibility are observed such that, C3H mice are considered semi-susceptible while BALB/c are considered susceptible. C3H mice develop a chronic disease in a high percentage of mice following MHV3 infection whereas BALB/c mice develop an acute and fatal hepatitis in 100 % of the mice. It has been speculated that the crucial factors in determining resistance or susceptibility are the expression of a monocyte derived monokine that demonstrates procoagulant activity (13, 14) , the antiviral state induced by IFN (4, 15, 16) and the virus replication in target cells (2, (17) (18) (19) .

IFN and macrophages are considered to be important elements in resistance against different viral infections (4, (20) (21) (22) . IFN-a/~ is readily induced during the initial phase of infection, well before the generation of an immune response, and IFN-y is rapidly induced following the generation of the immune response directed against the virus. Our previous studies, both in vitro and in vivo indicated that aT-cell dependent activation of macrophages in which IFN-y plays a major role is required to confer resistance to adult AI] mice against MHV3 (27) . These mice have macrophages that are very sensitive to IFN -y and are also sensitive to IFN-a/~. During infection, IFN-y is found in significant amounts in peritoneal exudate and serum and can be effective in restricting MHV3 multiplication in macrophages. On the other hand, mice from the susceptible BALBI c strain were shown to have macrophages that are not sensitive to IFN-a/~ or IFN-y. In spite of the high amounts of IFN-y detected in the serum and peritoneal exudate during the first days of infection, the macrophages could not restrict virus multiplication. The animals died 5 to 6 days after infection with the high amounts of IFN-y reflecting the virus replication and stimulation of the immune system (27) .

In the present work, we investigate the involvement the macrophage-IFN -y interaction in the age-dependent resistance showed by AI] mice after MHV3 infection.

Mice 15 and 60 days old AI] mice (originating from the Pasteur Institute, Paris, France, bred in our mouse colony and periodically controlled for the absence of Coronavirus or specific antibodies), here referred as young and adult AI] mice, respectively, were used to study the mortality, the virus growth and the IFN-aJ~ and -y synthesis in peritoneal exudate and serum after MHV3 infection. We also investigated the MHV3 growth and IFN-aJ~ synthesis in cultured peritoneal macrophages as well as the IFN-y synthesis in cultured spleen cells.

MHV3 was cultivated and titrated by plaque assay on L929 cells at 37°C as previously described (12) . Aliquots containing 2 x 10 5 plaque forming units per milliliter (PFU/ml) were stored at -80°C and used in all experiments. The MHV3 titers in tissues of infected animals or supernatants of cell cultures, obtained as described below, were expressed as PFU per milliliter of peritoneal exudate or supernatants (PFU/ml), or PFU per gram of liver (PFU/g).

The techniques used for preparation of the cell cultures have been described in detail elsewhere (12, 27) . Briefly, peritoneal exudate cells were collected by peritoneal lavage and cultured in RPMI 1640 containing 10 % FCS on 96 well plates at a concentration of 2 x 10 5 cells per well. After 2 h incubation they were washed three times to remove the nonadherent cells. Spleen cell suspensions were cultured at 5 x 10 6 cells per well in RPMI 1640 medium containing 10 % FCS on 24-well plates.

A cytopathic effect reduction test technique using monolayers of L 929 cells and encephalomyocarditis virus, described in detail in a previous paper (27) , was used as an IFN assay. For characterization of IFN-Ct/~ and IFN-y, antibodies to mouse IFN-aJ~ and monoclonal antibodies to recombinant mouse IFN-y were always used. These antibodies showed no cross-reactivity (27) .

After ip infection with 10 3 PFU of MHV3, all the young AI] mice were shown to be susceptible developing an acute hepatitis and dying 3 to 4 days after infection. On the other hand all the adult AI] mice were shown to be fully resistant, recovering from a mild disease 4 to 5 days after infection (data not shown).

The data shown in Figure 1 those obtained with adult AI] mice (Fig. 2) , where low titers of virus were found in the tissues and higher levels of IFN -y were found in the serum and peritoneal exudate.

The in vitro production of IFN-a/~, by cultured macrophages from normal young and adult AI] mice, stimulated with LPS (Fig. 3) , and the IFN -y produced by cultured spleen cells from normal and MHV3 immunized young and adult AI] mice, stimulated with Con A or MHV3 (Table 1) were investigated. In contrast to the results obtained with the cells from adult AI] mice, no synthesis of IFN-a/~ or IFN-y was detected in cultured cells from young AI] mice, indicating that these cells were not capable of producing IFN after specific or non-specific stimulation.

The results shown in Table 2 indicate that macrophages from young or adult AI] mice were very sensitive to the induction of an anti-MHV3 state by IFN-a/~ or IFN-y. No MHV3 replication occurred in the cultures from young AI] mice and very low virus titers were obtained in those from adult AI] mice. When the cells were activated with LPS (lipopolysaccharide from E. coli 0111 :B4-Difco Laboratories) only a slight inhibition of virus replication was observed in cultured cells from young AI] mice when. compared to the inhibition observed in cultured cells from adult AI] mice (Table 2 ). In a kinetic study, the macrophages were shown to be more sensitive to IFN-y than to IFN-a/B (data not shown). 

Previous work on MHV3 showed that both resistance gene( s) controlling the degree of viral replication in target cells such as macrophages, and an intact immune response are required for resistance of adult AI] mice to MHV3 infection (27) . We have demonstrated that the genetic dependent resistance of these mice to MHV3 depends on the expression of aT-cell dependent mechanism in which the sensitivity of macrophages to IFN-y plays a central role (27) .

In further support of the role of IFN-macrophage interaction as a crucial step for the resistance to MHV3, we have shown that a nutritionally induced hypercholesterolemia in resistant adult AI] mice caused susceptibility to MHV3 infection. The inhibition of host resistance was a consequence of an impairment of Kupffer cell sensitivity to IFN (29) .

Since resistance to MHV3 depends on the age and the genetic background of the animal, we decided to investigate whether or not the mechanism involved in the age-dependent resistance was of the same nature as that involved in the genetic-dependent resistance.

In contrast to the adult AI] mice, the data presented here clearly show that cultured spleen cells from young AI] mice were not capable of producing IFN-y in vitro (Table 1 ). Nor did young AI] mice infected with MHV3 show significant amounts of IFN-y in sera or peritoneal exudates, although the virus replicated to high titers, with the potential to elicit a strong stimulation of the immune system, as observed in the susceptible adult BALB/c mice (27) . Also, IFN-a/B were produced in very low levels in the course of the MHV3 infection ( Figs. 1 and 2) .

The peritoneal macrophages from young AI] mice, in contrast to macrophages from adult AI] mice, failed to synthesize detectable levels of IFN-alB after stimulation with LPS (Fig. 3) and consequently, only a slight inhibition of MHV3 replication, which was shown to be partially IFN -a/Bdependent (28) , was observed ( Table 2) . On the other hand, the exogenous IFN sensitivity of the macrophages from young AI] mice (Table 2 ) was comparable to that observed in the macrophages from adult AI] mice (27) .

Both types of IFN induced an anti-MHV3 state in these cells, although, as in macrophages from adult AI] mice (27) , lower doses of IFN-y were necessary to induce the antiviral state (data not shown).

The data presented here led us to the conclusion that the interaction of IFN and macrophage (mainly IFN-y) plays a central role in both age-and genetic-dependent resistance to MHV3 infection. In the case of age dependency, susceptibility of young AI] mice is caused by the immaturity of the immune system which is unable to synthesize IFN-y, whereas the genetic dependency relates to the inability of the BALB/c macrophages to respond to IFN activation (27) .

Our previous findings (27, 29) and these presented clearly indicate that the presence of IFN-y and the integrity of the macrophage functions are essential for resistance to MHV3 infection. They are intimately linked since the expression of the resistance is, to a large extent at least, a consequence of a T-cell-dependent mechanism of macrophage activation by IFN-y.

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Role of macrophages and interferon in natural resistance to mouse hepatitis virus type 3 infection

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Immunobiology of infection with herpes simplex virus

Interactions of herpesvirus with mononuclear phagocytes

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A major role of macrophage activation by interferon gamma during mouse hepatitis virus type 3 infection. 1. Genetically dependent resistance

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Increased susceptibility of mice to MHV3 infection induced by hypercholesterolemic diet: Impairment of Kupffer cell function

Laboratorio de Imunologia Viral