Specifically, our study presents evidence for the first time in amphibians that is consistent with the active and dynamic involvement of macrophage-like cells and NK cells in Xenopus adults during early stages of FV3 infection and before the onset of T-cell responses. In addition, our study provides evidence of the particular permissiveness of certain PLs to FV3 infection. Noticeably, the persistence of transcriptionally inactive FV3 genomic DNA in PLs may explain the occurrence of asymptomatic infection and suggests that FV3 is capable of covert infection.
Possible involvement of macrophages and NK cells at early stages of FV3 infection.
We have previously shown that during primary FV3 infection, it takes some time for the adult host's adaptive immune response to take place (24
). Both T-cell proliferation in the spleen and the infiltration of T cells in the kidney, the main site of infection, become significant only at 6 dpi and onward. Therefore, as in mammals, innate immune cell effectors are likely to play an important role at earlier stages of infection, as well as later on to orchestrate potent host adaptive immune responses. Mononucleated cells with multiple pseudopods typical of macrophage-like cells increase both in numbers and proportion in the peritoneal cavity as early as 1 day postinfection. This strongly suggests a faster response and preponderant role of this cell subset in the innate immune response against FV3. These macrophage-like cells show, especially at 2 and 3 dpi, obvious signs of activation including an increased size (i.e., higher forward scatter), numerous vacuoles, granules, phagocytic bodies, and a propensity to form aggregates. They become the major subset of PLs, constituting almost half of PLs at 6 dpi, the peak of primary FV3 infection. High numbers of PLs containing a majority (50 to 60%) of macrophage-like cells can be elicited by injection of heat-killed Escherichia coli
and can serve as efficient antigen-presenting cells for minor histocompatibility Ags (40
). Preliminary studies suggest that these bacterium-elicited PLs infected in vitro
with FV3 can induce proliferation of FV3-primed T cells and therefore are likely presenting viral Ags (31
). The accumulation of macrophage-like cells before and during the main T-cell response is consistent with their probable involvement as APCs in vivo
The widespread prevalence of FV3 (1
) and the fact that we have detected FV3 DNA in kidneys of a fraction of Xenopus
frogs from various suppliers (39
) raise the possibility that some of the frogs used in this study could have been exposed “naturally” to FV3 at some time prior to their being used. As mentioned in Materials and Methods, all animals used in the experiments reported here were either bred from our colony or purchased more than a year before the experiments were conducted. Importantly, regular screening for FV3 DNA in the kidneys by PCR in our colony has been negative so far. Tests for serum anti-FV3 antibodies by ELISA in our colony, including animals with primary infections, have also been negative (data not shown). Therefore, we think that one can reasonably consider these animals as immunologically naïve.
The overall similar but distinct increases in total PLs and accumulation of activated macrophages in the peritoneal cavity following a secondary FV3 infection provide further evidence that macrophages are critically involved in a typical host innate immune response during both primary and secondary FV3 infection. The earlier relative increase in the frequency of activated macrophages during secondary infection could be due to the presence of anti-FV3 antibodies and resulting immune complexes although almost nothing is known about macrophage activation by immune complexes in Xenopus.
In addition to the rapid cellular response of peritoneal macrophage-like cells initiated by FV3 infection, our study reveals a concomitantly increased expression of several genes involved in innate immune responses that, in mammals, are expressed by activated macrophages. The Xenopus
IL-1β gene homolog has been characterized and has been shown to be upregulated by lipopolysaccharide (LPS) (47
) or by heat-killed bacteria (25
). In addition, the functional characterization an IL-1β-like factor produced by stimulated peritoneal leukocytes has been reported in X. laevis
). Therefore, the upregulation of IL-1β induced by FV3 infection is consistent with the activation of innate immune cell effectors, especially macrophage-like cells. Although in mammals arginase 1 is a cytosolic enzyme, expressed almost exclusively in the liver, it is expressed by activated macrophages in mouse (34
) and is increasingly implicated in regulation of alternatively and classically activated macrophages (17
). We recently found a putative Xenopus
homolog of arginase 1 highly upregulated in a microarray analysis of splenocytes during an FV3 infection (H. D. Morales and J. Robert, unpublished data). We confirmed the homology of this gene by sequencing several X. laevis
expressed sequence tag (EST) clones (GenBank accession number NM_001086948). Phylogenetic analysis of the full-length deduced amino acid coding sequence of this gene unequivocally indicated its homology to arginase 1 (data not shown). The rapid induction of arginase 1 expression by FV3 infection together with the capacity for antigen presentation of PLs provides evolutionary evidence of the critical involvement of this enzyme in the activation process of macrophages. Furthermore, a TNF-α homolog has been characterized and has been shown to activate NF-κB (28
). The increased expression of this gene early during FV3 infection, therefore, is consistent with activation of macrophages. TNF-α is a cytokine produced by activated macrophages, T cells, and some other cells. It is a member of a group of cytokines that stimulate the acute-phase reaction in mammals and fish (13
). Recent studies in mammals suggest that TNF-α has strong antiviral effects. TNF-α is also involved in antiviral defense in invertebrates (6
). In the absence of specific Abs for Xenopus
macrophages, it is not possible to determine whether these cells are the main producers of TNF-α.
In addition to the possible involvement of macrophages, the present study clearly indicates an increase in NK cells in the peritoneal cavity during primary infection before the onset of the T-cell response in the spleen. NK cells are actively involved in host antiviral defenses in mammals (22
). Thus far, however, little is known about the antiviral capabilities of NK cells in amphibians. In Xenopus
, NK cells recognized by the 1F8 MAb are involved in antitumor responses and mediate cytotoxicity of MHC-negative target cells (18
). The increase in the number of 1F8-positive NK cells in PLs within the first week of infection, a time when antiviral innate immune responses are presumed to take place, suggests that NK cells are an important part of the first line of defense against FV3 infection. It remains to be determined if NK cells also infiltrate the kidney early during FV3 infection and, therefore, whether their increased numbers in the peritoneal cavity result from an increase in trafficking. The possible lack of NK cells at 3 and 6 days postsecondary infection would also merit further investigation.
The increase in IgM+
B cells at 6 dpi is interesting since we have reported that some B cells proliferate in the spleen during FV3 infection (24
) and that activated B cells expressing activation-induced cytidine deaminase are present in the periphery (26
). The drop in T-cell numbers at 3 dpi, on the other hand, is intriguing. Very little is known about the migration patterns of lymphocytes in Xenopus
. We have previously reported that total T cells accumulate as early as 3 dpi in the spleen, which in the absence of lymph node is the only draining lymphoid tissue in Xenopus
). Furthermore, splenic T-cell proliferation becomes significant only at 6 dpi, in parallel to the infiltration of CD8 T cells in the kidney, the main site of infection. Therefore, it is possible that the decrease in T cells in the peritoneal cavity at 3 dpi results from their drainage into the spleen, where they become activated. As such, the recovered T-cell population in PLs at 6 dpi may contain an increased fraction of activated T cells. Unfortunately, there are no good markers of T-cell activation in Xenopus
Collectively, our cellular and molecular data strongly suggest that a potent innate immune response mainly involving macrophage-like cells and NK cells is initiated upon primary FV3 infection in parallel with adaptive immune responses already described.
Apoptosis of PLs suggests FV3-mediated cytolysis.
We detected apoptotic bodies in PLs within the first days of infection. Apoptotic cells were more numerous within the first and third days of infection and significantly decreased by the sixth day. Pyknotic nuclei are indicative of late apoptosis, suggesting that susceptible cells are quickly eliminated upon FV3 infection and that the clearance by phagocytosis is either overwhelmed or delayed at this stage of infection. Although it can be argued that cells are being eliminated as a result of an active immune response (e.g., cell-mediated cytotoxicity), the fact that many apoptotic cells are seen so early upon infection suggests a direct cytolytic phase of FV3 infection in PLs. Apoptosis induced by FV3 has been reported (2
). Indeed, some infected cells detected by immunofluorescence microscopy with BG11 MAb were apoptotic, suggesting that active viral particles within the cells can induce cytotoxicity. Finally, it is still possible that some PL apoptosis induction is related to the release of TNF-α since its expression is enhanced during this period.
It would be interesting to determine whether some PLs are less susceptible to FV3-mediated apoptosis since it is often argued that permissive cells can act as viral reservoirs by allowing virus infection to occur without concomitant cytopathology (44
). In addition, phagocytic cells such as macrophages could acquire the virus from the apoptotic cells themselves (38
). Indeed, our observation of phagocytosis of apoptotic cells in the same cytospin preparations at 2 and 3 dpi suggests that apoptotic cells are quickly eliminated by the PLs. This is also consistent with the dynamic activity of PLs as they may be further activated by apoptotic stress signals.
Permissiveness of PLs to FV3 infection.
adults usually clear FV3 infection within 2 weeks, we have reported the occurrence in animals from various sources of asymptomatic FV3 carriers in which FV3 DNA was detected by PCR without corresponding symptoms of infection (39
). Given the involvement of macrophages in several cases of viral quiescence in mammals, we have postulated that macrophage-like cells may also be involved in FV3 covert infection in Xenopus
. Indeed, various viruses target macrophages in mammals for dissemination in the organism as well as to remain in a quiescent state (38
We have previously shown that PLs can be infected in vitro
and that a low level of FV3 infection can be detected in PLs in vivo
). In the present study, we further investigated the fate of FV3 infection in PLs. Interestingly, while we confirmed the ability of FV3 to infect and actively transcribe genes such as MCP and IE during the early phase of infection, the production of infectious particles was very poor compared to that occurring in the kidneys, and it was confined in a small fraction of PLs. Furthermore, the detection of viral DNA but no viral transcription at later stages of infection (15 and 21 dpi) suggests that FV3 can persist in a transcriptionally inactive form for some time in certain PLs. Although the fixative used for immunofluorescence study did not sufficiently preserve cell morphology to allow us to identify unequivocally which cell types were infected, cells resembling macrophage-like cells were observed. In addition, previous electron microscopy analysis has suggested that in addition to kidney tubular epithelium and, to a lesser extent, hepatocytes from heavily infected frogs, macrophage-like cells in these tissues might also be infected by FV3 (41
). Lastly, our TEM analysis of PLs infected in vitro
has revealed the presence of viral particles in the cytoplasm of cells with macrophage morphology. It will be interesting to determine in future experiments whether FV3 directly infects macrophage-like cells or if FV3 is acquired via phagocytosis of other infected cells. It is important to note that the particular pattern of infection by FV3 on PLs is observed with all frogs infected. In contrast, FV3 DNA is detected in only a fraction (8 to 30%) of asymptomatic animals from different sources and not purposely infected (39
). This suggests that the virus has the ability, under certain conditions that remain to be determined, to maintain itself without signs of infection. It is possible, for example, that long-term maintenance of FV3 depends on genetic factors segregating in the population.
Based on our finding that a fraction of Xenopus
frogs are asymptomatic FV3 carriers, we have proposed that X. laevis
could act as a viral reservoir, perhaps by maintaining a low level of infection in permissive cells in a covert fashion (39
). This suggests that FV3 can remain quiescent in PLs. Sublethal covert (not apparent) infections have been reported for several insect iridoviruses (45
). Although these viruses replicate without killing their host, they reduce host fitness. The possibility that RVs, such as FV3, are also capable of covert infections or quiescence would suggest that this phenomenon is a general attribute of iridoviruses.
Covert FV3 could be reactivated by stress signals such as a compromised immune system or subsequent infections. This could explain, for example, the report that RVs are also detected in a captive amphibian colony infected with chytrid fungus, Batrachochytrium dendrobatidis
). It is also possible that hormonal fluctuations, such as those induced by environmental toxins, could reactivate previously asymptomatic infections (5
). All of these possibilities need further testing, and with the use of PLs as a diagnostic tool in wild animals, the effect that emerging infectious diseases have on amphibian populations can be further investigated (32