ISG15 is critical in the control of Chikungunya virus infection independent of UbE1L mediated conjugation - PubMed (original) (raw)

ISG15 is critical in the control of Chikungunya virus infection independent of UbE1L mediated conjugation

Scott W Werneke et al. PLoS Pathog. 2011 Oct.

Abstract

Chikungunya virus (CHIKV) is a re-emerging alphavirus that has caused significant disease in the Indian Ocean region since 2005. During this outbreak, in addition to fever, rash and arthritis, severe cases of CHIKV infection have been observed in infants. Challenging the notion that the innate immune response in infants is immature or defective, we demonstrate that both human infants and neonatal mice generate a robust type I interferon (IFN) response during CHIKV infection that contributes to, but is insufficient for, the complete control of infection. To characterize the mechanism by which type I IFNs control CHIKV infection, we evaluated the role of ISG15 and defined it as a central player in the host response, as neonatal mice lacking ISG15 were profoundly susceptible to CHIKV infection. Surprisingly, UbE1L⁻/⁻ mice, which lack the ISG15 E1 enzyme and therefore are unable to form ISG15 conjugates, displayed no increase in lethality following CHIKV infection, thus pointing to a non-classical role for ISG15. No differences in viral loads were observed between wild-type (WT) and ISG15⁻/⁻ mice, however, a dramatic increase in proinflammatory cytokines and chemokines was observed in ISG15⁻/⁻ mice, suggesting that the innate immune response to CHIKV contributes to their lethality. This study provides new insight into the control of CHIKV infection, and establishes a new model for how ISG15 functions as an immunomodulatory molecule in the blunting of potentially pathologic levels of innate effector molecules during the host response to viral infection.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Serum concentration of IFN and IFN-induced chemokines is higher in CHIKV infected infants as compared to adults.

Patient sera were obtained from acute CHIKV infected adults (age = 16–86 years) and infants (age = 2–5 months). Age matched controls from La Réunion were tested. Chemokines / cytokines were measured using a Luminex assay. (A) IFNα and IFNγ levels; (B) IFN-induced molecules; and (C) Lymphocyte cell derived cytokines are shown. Whisker-box plots are shown (line indicates median; boxes represent first and third quartile; and bars define range). Mann-Whitney U-test was performed using a false discovery rate (FDR) procedure for generating corrected p-values. Comparisons were made between CHIKV patients versus their control cohort (in black); as well as adult versus infant patients (in red). ** indicates p<0.005, * indicates p<0.05. IFNα data from adult patients were previously reported , but are shown here for comparison to data from infected neonates.

Figure 2

Figure 2. Higher serum IFNα in infants is not explained by differences in viral load.

(A–C) Correlations between viral load vs. age; IFNα vs. age; and IFNα vs. viral load were evaluated. CHIKV infected infants are represented by blue squares; CHIKV infected adults are represented by red circles. Lines indicate linear regression and Spearman correlation (rs) values are shown. (D) Each group was divided into two subgroups (<median: low viral load or >median: high viral load; Median = 4.8×107) and statistic are represented (Mann Whitney test) (E) For analytes identified in Figure 1 that showed a correlation with viral load, IFN or age are plotted in a network array, illustrating the correlations identified in neonatal vs. adult individuals. Connecting lines indicate Spearman correlation (rs) values; positive correlations in red and negative correlations are depicted in blue. IFNα data from adult patients were previously reported , but are shown here for comparison to data from infected neonates.

Figure 3

Figure 3. Neonatal mice mount a robust IFN and pro-inflammatory cytokine/chemokine response following CHIKV infection.

Nine day old C57BL/6 mice were injected s.c. in the right flank with CHIKV. (A) The skin at the site of infection was harvested at the indicated times post infection and IFNβ, IRF7, Mx1 and ISG15 gene expression was evaluated by qRT-PCR. The ΔCt was calculated using GAPD as a reference gene. At various time points, sera was harvested and tested for (B) IFNα by ELISA and IFNγusing Luminex. (C) Serum cytokines and chemokines were also analyzed using Luminex. Each graph represents 2–3 independent experiments with 3 mice per experiment.

Figure 4

Figure 4. Endogenous Type I IFN is required for the control of neonatal CHIKV infection.

WT and IFNAR−/− mice were infected at 9 days of age with 2×105 PFU CHIKV s.c. (A) Mice were monitored for lethality for 21 days with data displayed as Kaplan-Meier curves. (B) Tissue and sera were collected on day 1 post-infection and viral titers were determined using a standard plaque assay. Mann-Whitney statistical comparison of WT and IFNAR−/− viral titers are shown where * indicates p<0.05, ** p<0.005 and *** p<0.0005, and vertical bars represent standard error of the mean.

Figure 5

Figure 5. Adjuvant induced type I IFN production protects neonates from CHIKV infection.

Mice (8 days of age) were injected i.p. with 25 µg of pIC and 24 hours later mice were infected s.c. in the right flank with CHIKV. (A) IFNα levels produced in the serum of neonatal mice 7 hours post injection with 25 µg pIC. (B) Survival was monitored daily for four weeks and displayed as Kaplan-Meier survival curves. (C) Mice were scored for clinical signs of disease on days 7, 13 and 18 post-infection as discussed in materials and methods. (D) IFNAR−/− mice (8 days of age) were injected i.p. with 25 µg of pIC and 24 hours later mice were infected with CHIKV s.c. Survival was monitored daily.

Figure 6

Figure 6. ISG15, independent of UbE1L, plays a critical role during the neonatal response to CHIKV.

(A–C) WT, UbE1L−/− and ISG15−/− mice were infected with 2×105 PFU CHIKV s.c. at nine days of age. (A) Mice were monitored for lethality for 21 days with data displayed as Kaplan-Meier curves. (B) Skin and muscle homogenates from the site of infection were collected on day 2 post infection and analyzed for ISG15 expression by western blot. (C) Sera collected on days 1, 3 and 6 post-infection and analyzed as in (B). (D and E) ISG15−/−, UbE1L−/− and WT mice (8 days of age) were injected i.p. with (D) 25 µg or (E) 10 µg of pIC and 24 hours later mice were infected s.c. with 2×105 PFU CHIKV. Survival was monitored daily for 21 days and displayed as Kaplan-Meier survival curves. Mice from all three genotypes that were pretreated with 25 µg of pIC and mock infected with PBS showed no lethality (data not shown).

Figure 7

Figure 7. ISG15−/− and UbE1L−/− mice display similar viral loads to WT mice.

Nine day old WT, UbE1L−/− and ISG15−/− mice were injected with 2×105 PFU CHIKV s.c. Tissue and sera were collected on days 1 (A) and 2 (B) post-infection and viral titers were determined by plaque assay; vertical bars represent standard error of the mean. A three way comparison of the three genotypes performed using a Kruskal-Wallis analysis was not significant except for the lung on day 2 post infection (*, where p<0.05).

Figure 8

Figure 8. ISG15−/− neonates display elevated cytokine levels during CHIKV infection.

Nine day old WT, IFNAR−/−, UbE1L−/− and ISG15−/− mice were injected with 2×105 PFU CHIKV s.c. (A) The skin at the site of infection was harvested and the expression of IFNβ mRNA was evaluated by qRT-PCR. Vertical bars represent mean with standard error of the mean. (B and C) Serum was collected at the indicated time post-infection. (B) IFNα levels were quantified using an ELISA, vertical bars represent mean with standard error of the mean. Statistical comparison between the three genotypes via Mann-Whitney and Kruskal-Wallis test was not significant for both (A) and (B). (C) Cytokine and chemokine levels were measured by Bioplex assay (Mann-Whitney * p<0.05, ** p<0.005). Vertical bars represent standard error of the mean.

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References

    1. Borgherini G, Poubeau P, Staikowsky F, Lory M, Le Moullec N, et al. Outbreak of chikungunya on Reunion Island: early clinical and laboratory features in 157 adult patients. Clin Infect Dis. 2007;44:1401–1407. - PubMed
    1. Lumsden WH. An epidemic of virus disease in Southern Province, Tanganyika Territory, in 1952-53. II. General description and epidemiology. Trans R Soc Trop Med Hyg. 1955;49:33–57. - PubMed
    1. Gerardin P, Guernier V, Perrau J, Fianu A, Le Roux K, et al. Estimating Chikungunya prevalence in La Reunion Island outbreak by serosurveys: two methods for two critical times of the epidemic. BMC Infect Dis. 2008;8:99. - PMC - PubMed
    1. Staikowsky F, Le Roux K, Schuffenecker I, Laurent P, Grivard P, et al. Retrospective survey of Chikungunya disease in Reunion Island hospital staff. Epidemiol Infect. 2008;136:196–206. - PMC - PubMed
    1. Mavalankar D, Shastri P, Raman P. Chikungunya epidemic in India: a major public-health disaster. Lancet Infect Dis. 2007;7:306–307. - PubMed

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