Functional Interferon System Is Required for Clearance of Lassa Virus

Abstract

Lassa virus (LASV) is the causative agent of Lassa hemorrhagic fever (LF) in humans, a deadly disease endemic to West Africa that results in 5,000 to 10,000 deaths annually. Here we present results demonstrating that functional type I and type II interferon (IFN) signaling is required for efficient control of LASV dissemination and clearance.

TEXT

Lassa virus (LASV), an arenavirus (family Arenaviridae), is enveloped and contains two single-stranded RNA segments of ambisense polarity encoding five proteins (1). LASV is the etiological agent of Lassa hemorrhagic fever (LF), a severe human disease that is reported in up to 100,000 patients annually in regions where it is endemic. Mortality varies between 5% and 10% for hospitalized patients (3). Individuals succumbing to LASV infection generate very limited or no anti-LASV humoral immune response, and histological examination of LF cases has revealed minimal immune cell infiltrates and tissue damage (14). These findings support the notion that morbidity and mortality associated with LASV infection are facilitated by the failure of the host's innate defense mechanisms to limit virus multiplication early during infection and to facilitate the initiation of an effective adaptive immune response capable of controlling and eliminating the infection. Accordingly, viremia level is a good predictor for the outcome of LF in patients (9). In addition, Lassa virus has been classified as a biosafety level 4 (BSL4) pathogen (3), making the scientific work with this pathogen more difficult.

Nonhuman primates and guinea pigs are two most commonly used animal models of LF (2, 6, 7, 12, 15). However, the use of these animal models is often associated with high costs, limited availability, and handling-safety risks. While most laboratory strains of mice are genetically resistant to LASV infection, mice with disrupted activation of major histocompatibility complex class I (MHC-I)-restricted CD8⁺ T cell response fail to control the infection and develop severe LF (5). At present, there is limited information on how innate immunity, especially the type I and II interferons (IFNs), contributes to the control of LASV infection. Therefore, we utilized mice lacking type I or both type I and II IFN receptors to investigate whether disruption of IFN signaling leads to impaired control of LASV infection in mice, as well as to establish an additional mouse model of LASV infection.

Mice that lack functional IFN signaling develop persistent infection after inoculation with LASV.

To this end, we infected 6- to 10-week-old male and female mice deficient for alpha/beta interferon receptor (IFNAR^−/−) (on the 129/Sv background; originally provided by Herbert Virgin at the Washington University School of Medicine, St. Louis, MO), mice deficient for alpha/beta and gamma interferon receptor (IFN-a/bgR^−/−) (provided by Chien-Te K. Tseng, University of Texas Medical Branch, Galveston, TX) (13), and control mice of the parental strain C57B6/129S1SvImJ (129S1/SvImJ) with 10⁴ PFU of LASV Josiah by the intraperitoneal (i.p.) route and monitored them for 25 days for survival and disease development. All animal procedures complied with the USDA guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Texas Medical Branch (UTMB). All infected animals were housed at BSL4.

All animals survived for at least 25 days after inoculation with LASV. Around day 11 p.i., all IFNAR^−/− mice developed disease characterized by ruffled fur and reduced activity, which lasted until the end of the study. We did not detect any signs of disease development in IFN-a/bgR^−/− mice infected with LASV. Mice in both groups experienced weight loss around day 11 p.i.; however, the magnitude and duration of weight loss in the IFNAR^−/− group were greater than those in the IFN-a/bgR^−/− group (Fig. 1A). By 17 days p.i., IFN-a/bgR^−/− mice infected with LASV recovered their weight and maintained it at the same level as the weight of uninfected IFN-a/bgR^−/− mice (Fig. 1A). In contrast, IFNAR^−/− mice infected with LASV did not fully recover their weight until the end of the study (Fig. 1A). Interestingly, we noticed that neither uninfected IFNAR^−/− nor IFN-a/bgR^−/− mice maintained their body weight at the same level as uninfected control 129S1/SvImJ mice (Fig. 1A). No fever was observed in any of the infected mice during the observation period (Fig. 1B).

Fig 1.

Changes in body weight and temperature of IFN receptor-deficient and immunocompetent mice after infection with LASV. IFNAR^−/−, IFN-a/bgR^−/−, and 129S1/SvImJ mice were inoculated with either 10⁴ PFU of LASV Josiah (n = 9) or sterile phosphate-buffered saline (PBS) (n = 9). Body temperature (A) and weight (B) change were recorded throughout the course of study. Average values and standard deviations are shown.

In order to assess the spread of LASV into the visceral organs and brains of infected mice, we humanely euthanized and necropsied three mice in each group and the titers of infectious virus in organ homogenates were analyzed by plaque assay (15). Both IFNAR^−/− mice lacking the type I IFN receptor and IFN-a/bgR^−/− mice lacking type I/II IFN receptors developed disseminated infection by day 11 p.i. (Fig. 2A). However, IFNAR^−/− mice were able to clear virus more efficiently than IFN-a/bgR^−/− mice, as indicated by a significant reduction of viral loads at day 25 p.i. in the spleen, lung, and liver and a mild reduction in the kidneys and heart of IFNAR^−/− mice compared to those of IFN-a/bgR^−/− mice (Fig. 2B). Notably, the virus titers in the brains of IFNAR^−/− and IFN-a/bgR^−/− mice were similar at 11 days p.i., and we observed only a mild reduction of titers in both groups on day 25 after infection.

Fig 2.

Organ virus titers and serum viremia in IFN receptor-deficient mice infected with LASV. Necropsy was performed on three LASV-infected mice in each group euthanized on day 11 (A) and 25 (B) p.i. Organs were homogenized and virus titers were determined by plaque assay. (C) Blood was collected at the indicated times, and virus titers in sera were determined by plaque assay. Horizontal dashes indicate average titer values. ND, not detected.

The results of organ titrations correlate with the ability of mice deficient for type I IFN receptor to better control viremia than mice that lack type I/II IFN receptors as indicated by gradual reduction of virus titer in the blood of IFNAR^−/− mice. Likewise, virus titers in the blood of IFN-a/bgR^−/− mice remained high up to 25 days p.i. (Fig. 2C). In contrast to IFN receptor-deficient mice, we were able to detect a very mild viremia in immunocompetent 129S1/SvImJ mice at 3 days p.i. that was cleared by day 11 after infection (Fig. 2C). Interestingly, neutralizing antibodies were below the limit of detection over the entire 25-day observation period (data not shown), which is in correlation with previous reports demonstrating delayed development of neutralizing antibody response in patients with LF and animals experimentally infected with LASV (4, 8, 9).

More prominent histopathological changes associated with LASV infection were detected in the brains and visceral organs of INFAR^−/− mice compared to those of IFN-a/bgR^−/− mice.

To compare pathological changes in mice deficient in type I (IFNAR^−/−) or both type I and II (IFN-a/bgR^−/−) IFN signaling, three randomly preselected animals from each group, as well as from the control group (129S1/SvImJ), were humanely euthanized 11 days p.i., and histological examinations were performed by a pathologist blinded to the genotypes of the animals. Below we describe the histopathological changes that we observed in the brains and visceral organs of LASV-infected mice, with the exception of the heart, where we did not detect pathological changes in any of the experimental animals (data not shown).

The brains of LASV-infected IFNAR^−/− mice showed mild diffuse cerebral leptomeningitis with infiltration of mononuclear cells (MNCs). Also, rare perivascular cuffs (1 to 2 cells thick) of MNCs and occasional endothelial hypertrophy were observed in the cortex (Fig. 3B). In contrast, we only rarely observed foci of leptomeningitis and one-cell-thick perivascular cuffs of infiltrating lymphocytes in the brains of IFN-a/bgR^−/− mice (Fig. 3C).

Fig 3.

Comparative histopathology of mice infected with LASV. IFNAR^−/−, IFN-a/bgR^−/−, and 129S1/SvImJ mice were infected with 10⁴ PFU of LASV Josiah via the i.p. route or mock infected. Representative hematoxylin-and-eosin-stained tissue sections from animals euthanized on day 11 p.i. are shown. Magnifications: ×20 (brain, lung, liver, and kidney sections) and ×10 (spleen sections).

The lungs of IFNAR^−/− and IFN-a/bgR^−/− mice showed diffuse peribronchial MNC infiltration (5 to 10 cells thick) involving >75% of airway profiles. Areas of focal alveolar edema and perivascular inflammation were also detected (Fig. 3E and F).

The livers of IFNAR^−/− mice infected with LASV showed mild periportal inflammation associated with infiltration of lymphocytes and plasma cells. However, no lobular inflammation or apoptosis/necrosis was seen (Fig. 3H). The livers of IFN-a/bgR^−/− mice contained MNC infiltrates in some portal triads and rare foci of lobular inflammation (Fig. 3I).

Histopathological changes observed in the spleens of IFNAR^−/− mice were characterized by expansion of the white pulp that appeared poorly organized. However, germinal centers were clearly seen in all of the examined organs. Red pulp was congested and moderately cellular (Fig. 3K). The spleens of the IFN-a/bgR^−/− mice showed distinct white pulp with large active germinal centers. However, red pulp cellularity was increased (Fig. 3L).

The kidneys of IFNAR^−/− mice showed scattered small foci of interstitial inflammation, either expanding the interstitium or focally forming perivascular nodular aggregates of lymphocytes (Fig. 3N). In contrast, histological observation of the kidneys of IFN-a/bgR^−/− mice revealed rare scattered subtle interstitial lesions of inflammation without expansion in only one animal out of three examined (Fig. 3O).

Only mild inflammatory changes were detected in the brains and visceral organs of LASV-infected control 129S1/SvImJ mice (Fig. 3A, D, G, J, and M).

In summary, we present here for the first time that mice lacking type I or both type I and II IFN receptors developed disseminated nonlethal infection with LASV. In our previous studies with JUNV, in contrast to LASV, infected IFN receptor knockout (KO) mice developed severe disease and lethality (10). This may be due to the fact that, according to our observations, LASV is less cytopathic than JUNV and, therefore, may be inducing less direct cell and organ damage in these mice. Our data presented here correlated with the results from previously published experiments obtained in MHC-I-deficient mice (5) that demonstrated the important role of T cells in the clearance of the virus. Based on these data, we hypothesized that the lack of IFN signaling might have a similar effect on virus clearance, as it clearly impacts the ability of the host to upregulate MHC-I molecules. Accordingly, we performed experiments described in this paper by utilizing infection of immunodeficient mice with LASV. Our experimental data strongly suggest that both type I and type II IFN pathways are essential for virus clearance. The ability of type I IFN receptor KO mice to clear virus more efficiently than type I/II IFN receptor KO mice can potentially be explained by strongly impaired activation of CD8⁺ T cells via IFN-γ-mediated mechanism(s). Interestingly, a recent study showed that type I IFN-dependent induction of peritoneal IFN-γ production by natural killer (NK) cells in mice inoculated with lymphocytic choriomeningitis virus (LCMV) by the i.p. route led to enhanced resistance to viral infection (11). These data suggest that type I IFN may have a synergistic effect on IFN-γ-mediated immune responses to arenavirus infection. Accordingly, these results are in agreement with the observation of more prominent pathological changes in the organs of IFNAR^−/− mice; however, development of the early immune response and virus clearance required the presence of functional type I and type II IFN signaling. Higher organ titers did not correlate with pathological changes, as was shown for human cases of LF (14). This phenomenon can also be explained by the impaired CD8⁺ T cell-mediated cytotoxic response in both KO mouse strains used in the current study. Further experiments to evaluate the role of type I and II IFN in the induction and maintenance of CD8⁺ T cells in response to LASV need to be considered. However, mice deficient in production of type I and type II IFN receptors that develop persistent disseminated infection in the visceral organs and brain may become a valuable tool to study LASV infection in immunocompromised individuals as well as neurological complication during and/or after LASV infection. Additionally, these may become a useful animal model for in vivo screening of antiviral drugs against LASV that directly target the virus life cycle and do not require the involvement of host IFN pathways.