This site needs JavaScript to work properly. Please enable it to take advantage of the complete set of features!
Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log in
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Dec 15;13(12):e1006748.
doi: 10.1371/journal.ppat.1006748. eCollection 2017 Dec.

Inflammatory monocytes mediate control of acute alphavirus infection in mice

Affiliations

Inflammatory monocytes mediate control of acute alphavirus infection in mice

Kelsey C Haist et al. PLoS Pathog. .

Abstract

Chikungunya virus (CHIKV) and Ross River virus (RRV) are mosquito-transmitted alphaviruses that cause debilitating acute and chronic musculoskeletal disease. Monocytes are implicated in the pathogenesis of these infections; however, their specific roles are not well defined. To investigate the role of inflammatory Ly6ChiCCR2+ monocytes in alphavirus pathogenesis, we used CCR2-DTR transgenic mice, enabling depletion of these cells by administration of diptheria toxin (DT). DT-treated CCR2-DTR mice displayed more severe disease following CHIKV and RRV infection and had fewer Ly6Chi monocytes and NK cells in circulation and muscle tissue compared with DT-treated WT mice. Furthermore, depletion of CCR2+ or Gr1+ cells, but not NK cells or neutrophils alone, restored virulence and increased viral loads in mice infected with an RRV strain encoding attenuating mutations in nsP1 to levels detected in monocyte-depleted mice infected with fully virulent RRV. Disease severity and viral loads also were increased in DT-treated CCR2-DTR+;Rag1-/- mice infected with the nsP1 mutant virus, confirming that these effects are independent of adaptive immunity. Monocytes and macrophages sorted from muscle tissue of RRV-infected mice were viral RNA positive and had elevated expression of Irf7, and co-culture of Ly6Chi monocytes with RRV-infected cells resulted in induction of type I IFN gene expression in monocytes that was Irf3;Irf7 and Mavs-dependent. Consistent with these data, viral loads of the attenuated nsP1 mutant virus were equivalent to those of WT RRV in Mavs-/- mice. Finally, reconstitution of Irf3-/-;Irf7-/- mice with CCR2-DTR bone marrow rescued mice from severe infection, and this effect was reversed by depletion of CCR2+ cells, indicating that CCR2+ hematopoietic cells are capable of inducing an antiviral response. Collectively, these data suggest that MAVS-dependent production of type I IFN by monocytes is critical for control of acute alphavirus infection and that determinants in nsP1, the viral RNA capping protein, counteract this response.

PubMed Disclaimer

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Ly6Chi monocytes and NK cells are depleted from DT-treated CCR2-DTR mice.
(A, B, C) WT (n = 6–7 mice/group) or CCR2-DTR (n = 5–6 mice/group) C57BL/6 mice were inoculated in the left rear footpad with (A) PBS, (B) RRV-T48, or (C) CHIKV. At days -1 and +2 relative to infection, mice were i.p. administered DT. 24 h after the last DT administration, the frequency of Ly6Chi monocytes (Ly6ChiCD11b+CD43+Ly6G-), NK cells (NK1.1+CD11b+Ly6C-Ly6G-), and neutrophils (Ly6G+CD11b+CD43+Ly6C+) in the blood were determined by flow cytometry. Data are pooled from two independent experiments. P values were determined by Student’s t-test. *, P < 0.05; ***, P < 0.001. (D) WT (n = 4–5 mice/group) or CCR2-DTR (n = 2–5 mice/group) C57BL/6 mice were inoculated in the left rear footpad with either PBS or RRV-T48. DT was administered as described for A-C. At 48 h after the last DT administration, the number of NK cells (NK1.1+CD11b+Ly6C-Ly6G-), neutrophils (Ly6G+CD11b+CD43+Ly6C+), and various Ly6C+CD11b+ and Ly6ChiCD11b+ myeloid subsets were determined by flow cytometry. Data are pooled from two independent experiments. P values were determined by one-way ANOVA with a Tukey’s multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 2
Fig 2. Depletion of CCR2+ cells enhanced viral loads in tissues and the severity of acute RRV infection.
WT or CCR2-DTR C57BL/6 mice were inoculated in the left rear footpad with 1,000 PFU of RRV-T48 or RRV-T48-nsP16M. At days -1 and +2 relative to infection, mice were i.p. administered DT. 1 h prior to virus infection, Ly6Chi monocytes were adoptively transferred into a subset of CCR2-DTR mice. At 5 dpi, viral RNA levels in (A) skeletal muscle tissue and (B) contralateral ankle tissue were quantified by qRT-PCR. (C) At 5 dpi, infectious virus in the serum was quantified by plaque assay. (D) The percent starting body weight was determined daily (n = 6–17 mice/group). Data are pooled from three independent experiments. P values were determined by one-way ANOVA with a Tukey’s multiple comparison test (A-C) and a repeated measures two-way ANOVA with a Bonferroni’s multiple comparison test (D). *, P <0.05; **, P < 0.01; ***, P < 0.001.
Fig 3
Fig 3. Antibody-mediated depletion of Ly6Chi monocytes exacerbates acute RRV infection.
WT C57BL/6 mice were administered (A and B) anti-NK1.1 (n = 4) or a control antibody (n = 4), (C and D) anti-Gr1 (n = 8) or a control antibody (n = 8), or (E and F) anti-Ly6G (n = 4) or a control antibody (n = 4) at day -1 and day +2 relative to infection with the indicated viruses. At 5 dpi, (A, C, E) viral RNA levels in skeletal muscle tissue were quantified by qRT-PCR, and (B, D, F) the percent starting body weight was determined daily. Data are from one (anti-NK1.1 and anti-Ly6G) or two (anti-Gr1) independent experiments. P values were determined by one-way ANOVA with a Tukey’s multiple comparison test (A, C, E) or a repeated measures two-way ANOVA with a Bonferroni’s multiple comparison test (B, D, F). *, P <0.05; **, P < 0.01; ***, P < 0.001.
Fig 4
Fig 4. Depletion of CCR2+ cells enhances pathogenicity of the attenuated RRV-T48-nsP16M mutant virus.
WT or CCR2-DTR C57BL/6 mice (n = 3–7 mice/group) were inoculated in the left rear footpad with 1,000 PFU of RRV-T48 or RRV-T48-nsP16M. At days -1 and +2 relative to infection, mice were i.p. administered DT. (A) The percent starting body weight and (B) musculoskeletal disease score were determined daily. (C) At 10 dpi, viral RNA levels in skeletal muscle tissue were quantified by qRT-PCR. P values were determined by a repeated measures two-way ANOVA with a Bonferroni’s multiple comparison test (A, B) and by one-way ANOVA with a Tukey’s multiple comparison test (C) *, P <0.05; **, P < 0.01; ***, P < 0.001.
Fig 5
Fig 5. Depletion of CCR2+ cells enhanced viral loads in tissues and the severity of disease during acute CHIKV infection.
WT (n = 9) or CCR2-DTR (n = 6) C57BL/6 mice were inoculated in the left rear footpad with 1,000 PFU of CHIKV. At days -1 and +2 relative to infection, mice were i.p. administered DT. (A) The percent starting body was determined daily. At 5 dpi, (B) viral RNA levels in the contralateral ankle were quantified by qRT-PCR and (C) infectious virus in the serum was quantified by plaque assay. Data are pooled from three independent experiments. P values were determined by a repeated measures two-way ANOVA with a Bonferroni’s multiple comparison test (A) and by Student’s t-test (B, C). *, P <0.05; **, P < 0.01; ***, P < 0.001.
Fig 6
Fig 6. MAVS-dependent induction of type I IFN in monocytes during RRV infection.
(A) IFNα2 mRNA expression levels in enriched WT bone marrow monocytes co-cultured for 18 h with uninfected, RRV-T48- or RRV-T48-nsP16M-infected-infected Vero cells (n = 6/group). (B) IFNα2 mRNA expression levels in enriched WT or Irf3-/-;Irf7-/- bone marrow monocytes co-cultured for 18 h with uninfected or RRV-T48-infected-infected Vero cells (n = 6/group). (C) IFNα2 mRNA expression levels in enriched WT or Mavs-/- bone marrow monocytes co-cultured for 18 h with uninfected or RRV-T48-infected-infected Vero cells (n = 6-9/group). Data are normalized to 18S rRNA levels and are expressed as the relative expression (n-fold increase) over expression in uninfected Vero cells without monocytes. Data are combined from at least two independent experiments. P values were determined by one-way ANOVA with a Tukey’s multiple comparison test. *, P <0.05; **, P < 0.01; ***, P < 0.001.
Fig 7
Fig 7. Induction of type I IFN expression in monocytes requires virus infection.
(A-B) Enriched WT bone marrow monocytes were co-cultured with Vero cells infected with RRV-GFP (n = 3/group). After 18 h of co-culture, GFP expression in Vero cells and monocytes was measured by flow cytometry. (A) Shown are representative histograms. (B) The percent GFP+ monocytes and Vero cells. Data are representative of two independent experiments. (C-D) Enriched WT bone marrow monocytes were co-cultured with uninfected or RRV-GFP-infected Vero cells in the presence or absence of 1 μM Latrunculin B. After 18 h of co-culture, (C) IFNα2 mRNA expression level in monocytes was quantified by qRT-PCR. Data are normalized to 18S rRNA levels and are expressed as the relative expression (n-fold increase) over expression in uninfected Vero cells without monocytes. (D) The percent GFP+ Vero cells and monocytes were measured by flow cytometry based on GFP expression within the CD45- Vero cells and the CD45+ monocytes. Data are combined from two independent experiments. P values were determined by one-way ANOVA with a Tukey’s multiple comparison test.
Fig 8
Fig 8. Inflammatory monocytes express Irf7 and are viral RNA-positive during RRV infection in vivo.
WT C57BL/6 mice were inoculated in the left rear footpad with PBS or 1,000 PFU of RRV-T48 or RRV-T48-nsP16M. At 5 dpi, Ly6Chi monocytes were enriched from the bone marrow or FACS-sorted from the blood and from enzymatically-digested muscle tissue. (A-C) Representative flow cytometry gating of cells sorted from the (A) bone marrow, (B) blood of RRV-infected mice, and (C) skeletal muscle of RRV-infected mice. (D) Irf7 mRNA expression levels in enriched cell populations were quantified by qRT-PCR. Data are normalized to 18S rRNA levels and are expressed as the relative expression (n-fold increase) over expression in Ly6Chi bone marrow monocytes from uninfected mice. Each data point represents monocytes sorted from the combined tissues of 3 mice. Each bar represents the arithmetic mean of two independent experiments. (E-F) RRV RNA levels in enriched monocyte/macrophage populations sorted from bone marrow of uninfected mice or skeletal muscle tissue of RRV-infected mice were quantified by qRT-PCR. (E) Each data point represents monocytes sorted from the combined muscle tissues of 3 mice. (F) Each data point represents monocytes/macrophages sorted from the muscle tissue of 2 mice (5 dpi) or a single mouse (7 dpi).
Fig 9
Fig 9. Viral loads of RRV-T48 and RRV-T48-nsP16M are equivalent in Mavs-/- mice.
WT (n = 7/group) or Mavs-/- (n = 10/group) C57BL/6 mice were inoculated in the left rear footpad with 1,000 PFU of RRV-T48 or RRV-T48-nsP16M. At 5 dpi, viral RNA levels in skeletal muscle tissue were quantified by qRT-PCR. Data are pooled from three independent experiments. P values were determined by one-way ANOVA with a Tukey’s multiple comparison test. **, P < 0.01; ***, P < 0.001.
Fig 10
Fig 10. CCR2+ hematopoietic cells protect Irf3-/-;Irf7-/- mice from severe RRV infection.
Irf3-/-;Irf7-/- C57BL/6 mice (n = 5/group) were lethally irradiated and reconstituted with Irf3-/-;Irf7-/- or CCR2-DTR bone marrow. At 6 weeks post-irradiation, mice were inoculated in the left rear footpad with 10 PFU of RRV-T48-nsP16M. At days -1 and +2 relative to infection, mice were i.p. administered PBS or DT. Mice were monitored daily for (A) morbidity and (B) mortality. P values were determined by two-way ANOVA with a Bonferroni’s multiple comparisons test. ***, P < 0.001. At 7 dpi, (C) viral RNA levels in skeletal muscle tissue were quantified by qRT-PCR and P values were determined by an unpaired t-test. ***, P < 0.001.

References

    1. Suhrbier A, Jaffar-Bandjee MC, Gasque P. Arthritogenic alphaviruses—an overview. Nat Rev Rheumatol. 2012;8(7):420–9. Epub 2012年05月09日. doi: 10.1038/nrrheum.2012.64 . - DOI - PubMed
    1. Weaver SC, Lecuit M. Chikungunya virus and the global spread of a mosquito-borne disease. N Engl J Med. 2015;372(13):1231–9. doi: 10.1056/NEJMra1406035 . - DOI - PubMed
    1. Leparc-Goffart I, Nougairede A, Cassadou S, Prat C, de Lamballerie X. Chikungunya in the Americas. Lancet. 2014;383(9916):514 Epub 2014年02月11日. doi: 10.1016/S0140-6736(14)60185-9 . - DOI - PubMed
    1. Petersen LR, Powers AM. Chikungunya: epidemiology. F1000Res. 2016;5 doi: 10.12688/f1000research.7171.1 . - DOI - PMC - PubMed
    1. Morrison TE. Reemergence of chikungunya virus. J Virol. 2014;88(20):11644–7. doi: 10.1128/JVI.01432-14 . - DOI - PMC - PubMed

Publication types

MeSH terms

Full text links
Public Library of Science full text link Public Library of Science Free PMC article
Cite

AltStyle によって変換されたページ (->オリジナル) /