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The host inflammatory response contributes to disease severity in Crimean-Congo hemorrhagic fever virus infected mice [1]

['Joseph W. Golden', 'Virology Division', 'United States Army Medical Research Institute Of Infectious Diseases', 'Fort Detrick', 'Maryland', 'United States Of America', 'Xiankun Zeng', 'Pathology Division', 'Curtis R. Cline', 'Jeffrey M. Smith']

Date: 2022-07

MAVS-dependent signaling is required for CCHFV-mediated acute disease in IFN-I blockaded mice

Because MAVS is important in the IFN-I response, we initially investigated if it alone played an important role in host susceptibility to CCHFV infection. Mice lacking MAVS (MAVS KO), but with otherwise intact IFN-I signaling, were not susceptible to CCHFV infection by the murine lethal strain Afg09-2990 and exhibited no weight loss or mortality. This was in contrast to the extensive weight loss and 80% mortality in CCHFV strain Afg09-2990 infected non-transgenic wild-type (WT) mice (C57BL/6) treated with a monoclonal antibody (mAb)-5A3 to block IFN-I signaling (Fig 1A). This finding indicated that loss of MAVS does not enhance susceptibility of mice to CCHFV. Because MAVS induces inflammatory signaling pathways independent of IFN-I activation [32,33], we next evaluated CCHFV infection in MAVS-deficient mice in the presence of an antibody-induced IFN-I signaling blockade. IFN-I antibody blocked and infected MAVS KO mice lost weight starting on day 2 and continued decreasing until day 9 before recovering. (Figs 1B and S1). Weight loss was similar to IFN-I blocked and infected WT mice (B6:129). Despite weight loss, all MAVS KO mice survived infection, whereas all WT mice succumbed to disease by day 6. CCHFV infection in IFN-I blockaded mice was repeated in 19 MAVS KO mice over multiple studies with universal survival, contrasting against 100% mortality in control WT mice (Log-rank; p<0.0001) (Figs 1B and 1E and S1).

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TIFF original image Download: Fig 1. MAVS KO mice with IFN-I signaling blocked are protected against lethal infection by CCHFV. A. C57BL/6 mice or MAVS KO mice (n = 5/group) were infected with CCHFV and survival and weight loss were monitored and plotted using Prism software. Only WT mice were treated with MAb-5A3 24h after infection to block IFN-I. IFN-I was not blocked by antibody in the MAVS KO mice. B. B6.129 or MAVS KO mice (n = 6/group) were infected with CCHFV and both groups treated 24 h post-infection were treated with mAb-5A3. Survival and weight loss were monitored for 20 days and plotted using Prism software. Survival significance was determined by log-rank analysis; ***p<0.0001. C. Viral RNA in serum and liver was examined on day 4, 10 and 15 by RT-qPCR (n = 3 per group). Mean titers +/- SEM of the estimated PFUs (PFUe) were graphed. The dashed black line denotes limit of detection. Statistical significance was determined by one-way ANOVA; *p<0.05. D. Monocyte chemoattractants and inflammatory cytokines were measured from the serum (n = 3 per group) of CCHFV infected mice on day 4 using a multiplex system. Statistical significance compared to uninfected controls was determined by one-way ANOVA; *p<0.05, ***p<0.001. E. C57BL/6, MDA5 KO or MAVS KO mice (n = 8 per group) were infected with CCHFV and 24 h post-infection were treated with MAb-5A3. Survival and weight loss were monitored for 20 days. Significance determined by log-rank analysis (p<0.0001). https://doi.org/10.1371/journal.ppat.1010485.g001

Levels of viral genome in the liver and serum on day 4 post-infection were reduced in mAb-5A3 treated MAVS KO mice compared to WT mice treated with mAb-5A3 (Fig 1C). The difference in viral load was statistically significant in the liver, but not serum (one-way ANOVA; p<0.05). Viral RNA was also detected on day 10, and to a lesser extent on day 15 in both the liver and serum of MAVS KO animals. MAVS deficient mice had a large reduction in the cytokines TNF-α, IL-6, IFN-γ, IL-18 and the chemokines CCL7 and CCL2 on day 4 compared to the infected WT mice (Fig 1D). MAVS signaling is transduced via MDA5 and RIG-I. However IFN-I blocked, CCHFV-infected MDA5 deficient animals did not have a survival advantage over control mice (Fig 1E). These findings indicated that loss of MAVS, but not MDA5, confers a significant survival advantage to CCHFV infected mice in the absence of IFN-I signaling.

The liver is a key target organ of CCHFV in humans and in mice [3,9,19,34]. Histopathological changes in livers from CCHFV infected WT (B6.129) and MAVS KO mice in the presence of IFN-I signaling blockade were evaluated on day 4 for both strains of mice and day 10 and 15 for MAVS KO mice. On Day 4, WT mice developed hepatic lesions, marked by extensive inflammation with hepatocellular degeneration and necrosis (Fig 2). Kupffer cell hypertrophy was evident in the sinusoids, and occasional periportal oval cell hyperplasia. In contrast, liver pathology was marginal on day 4 in MAVS KO mice and was characterized by minimal multifocal inflammation. Liver pathology was more prevalent in MAVS KO mice on day 10, with increased inflammation and moderate hepatocelluar necrosis. However, these hepatic lesions were relatively mild in comparison to the damage observed in infected, WT mice on day 4. By day 15, liver injury is predominantly absent in MAVS KO animals, with only minimal inflammation detected (Fig 2B).

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TIFF original image Download: Fig 2. Hepatic injury is reduced in CCHFV infected MAVS KO mice. A. Representative H&E staining of livers from WT (Day 4) and MAVS KO mice infected with CCHFV in the presence of IFN-I blockade harvested on Day 4 and 10 (n = 3/group/time point). mAb-5A3 treated Day 4 B6.129 mice show increased liver pathology compared to MAVS KO mice or uninfected mice. Inflammation and necrosis were increased in the liver on day 10 in the MAVS KO mice, compared to the day 4 in the MAVS KO mice, but pathology was still less severe than in the WT (B6.129) mice. B. Pathology scores for WT (Day 4) or MAVS KO mice (Day 4, 10 and 15) were plotted for the indicated lesions (n = 3 per group). Statistical significance of infection in MAVS KO mice compared to infected controls was determined by one-way ANOVA; **p<0.05, ***p<0.001 or NS; not significant. C Representative ISH staining of livers. ISH stained tissue was counterstained with hematoxylin. D. Liver sections from infected (WT [B6.129] or MAVS KO) or uninfected WT (B6.129) mice were stained with anti-CLEC4F (red) and anti-CCHFV N protein antibodies (green). Cell nuclei were stained with DAPI. E and F. IFA demonstrates increased number of CD68+ macrophages (E, green) and Ki67+ proliferating cells (E, red) or MPO+ neutrophil granulocytes (F, green) and CD45+ leukocytes (F, red) in livers of WT (B6.129) mice compared to MAVS KO or uninfected mice. Nuclei are stained with DAPI (blue). https://doi.org/10.1371/journal.ppat.1010485.g002

In situ hybridization (ISH) showed viral genomic RNA was prevalent in WT mouse livers on day 4, but the level of staining was comparatively reduced in MAVS KO mice on day 4 and day 10 (Fig 2C). The liver ISH staining pattern suggested that Kupffer cells were the primary cells targeted by CCHFV in the MAVS KO mice on day 4. We previously reported that on day 4 infected WT mice lacking IFN-I activity have nearly a complete loss of Kupffer cells indicated by an absence of CLEC4F+ staining [19]. Here, WT mice also had an extensive loss of CLEC4F+ staining on day 4 (Fig 2D). In contrast, Kupffer cell loss was absent in infected MAVS KO mice with levels of CLEC4F+ staining similar to uninfected mice. In MAVS KO mice, viral nucleocapsid (N) protein was predominantly localized to Kupffer cells on day 4. N protein staining of MAVS KO mice became more disseminated on day 10, present in CLEC4F positive and negative cells. At the day 4 time point, an influx of CD68+ monocytes/macrophages and Ki67+ proliferating cells and an increase in myleoperoxidase (MPO)+ neutrophil granulocytes and CD45+ leukocytes in the WT animals was detected (Fig 2E and 2F). Staining of MPO+ neutrophil granulocytes was reduced in MAVS mice on day 4, but higher than uninfected mice. Staining of Ki67+ proliferating cells and CD45+ leukocytes in MAVS KO mice was similar to uninfected animals. However, CD68+ staining was still present in MAVS KO mice, but the staining pattern was more indicative of Kupffer cells which are CD68+. These results demonstrated that the loss of MAVS ameliorates CCHFV-induced hepatic inflammation.

Gene expression profiling of 297 genes associated with host immunology and inflammatory processes was examined using the NanoString nCounter platform and total RNA isolated from mouse liver homogenates (n = 3/time points) on day 4 (WT, MAVS KO and uninfected WT mice) and day 10 (MAVS KO). The three groups of animals (infected WT, MAVS KO and uninfected WT mice, all IFN-I blocked) had bulk transcript profiles that were uniquely distributed by principal component (PC) analysis (Fig 3A). MAVS KO day 4 and day 10 groups mostly clustered together with one exception, but uninfected and infected WT mice clustered separately. Transcriptomic profiling demonstrated that on day 4 MAVS KO infected mice have blunted inflammatory responses compared to WT, infected mice. On day 10, some inflammatory response gene signatures are restored to levels similar to those of infected WT mice (Fig 3B). In day 4 samples, pathway scoring using Ingenuity Pathway Analysis (IPA) from QIAGEN indicated decreases in gene signatures for viral pathogenesis, death receptor signaling, IL-6 signaling, and NF-kB activation by viruses in MAVS KO livers (Fig 3C and 3D). In the MAVS KO group, several pathways were not elevated above non-infected animals, including IL-6, HMGB1, and iNOS; however, by day 10 Z-scores for those pathways were similar to infected WT mice on day 4 (S2 Fig). These findings indicated that MAVS deficiency delays inflammatory responses in CCHFV infected mice compared to WT animals.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010485

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