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. 2010 Oct 19;1(4):e00219-10.
doi: 10.1128/mBio.00219-10.

Autocrine interferon priming in macrophages but not dendritic cells results in enhanced cytokine and chemokine production after coronavirus infection

Haixia Zhou  1 , Jincun Zhao, Stanley Perlman
Affiliations

Autocrine interferon priming in macrophages but not dendritic cells results in enhanced cytokine and chemokine production after coronavirus infection

Haixia Zhou et al. mBio. .

Abstract

Coronaviruses efficiently inhibit interferon (IFN) induction in nonhematopoietic cells and conventional dendritic cells (cDC). However, IFN is produced in infected macrophages, microglia, and plasmacytoid dendritic cells (pDC). To begin to understand why IFN is produced in infected macrophages, we infected bone marrow-derived macrophages (BMM) and as a control, bone marrow-derived DC (BMDC) with the coronavirus mouse hepatitis virus (MHV). As expected, BMM but not BMDC expressed type I IFN. IFN production in infected BMM was nearly completely dependent on signaling through the alpha/beta interferon (IFN-α/β) receptor (IFNAR). Several IFN-dependent cytokines and chemokines showed the same expression pattern, with enhanced production in BMM compared to BMDC and dependence upon signaling through the IFNAR. Exogenous IFN enhanced IFN-dependent gene expression in BMM at early times after infection and in BMDC at all times after infection but did not stimulate expression of molecules that signal through myeloid differentiation factor 88 (MyD88), such as tumor necrosis factor (TNF). Collectively, our results show that IFN is produced at early times postinfection (p.i.) in MHV-infected BMM, but not in BMDC, and primes expression of IFN and IFN-responsive genes. Further, our results also show that BMM are generally more responsive to MHV infection, since MyD88-dependent pathways are also activated to a greater extent in these cells than in BMDC.

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Figures

FIG 1
FIG 1
Mouse hepatitis virus (MHV)-infected bone marrow-derived macrophages (BMM) produce type I IFN at late times p.i. (A) BMM or bone marrow-derived dendritic cells (BMDC) were infected with MHV or Sendai virus (SenV), and IFN protein levels were determined using a VSV-based IFN bioassay at the indicated times (in hours) postinfection (p.i. ). BMM-MHV, BMM infected with MHV; BMDC-MHV, BMDC infected with MHV. (B) MHV-induced IFN production in panel A is shown using an enlarged scale. (C) BMM were infected with MHV or SenV, and IFN-β mRNA levels were measured by real-time qPCR. Cycle threshold (CT) values were calculated as a ratio to hypoxanthine phosphoribosyltransferase (HPRT) as described in Materials and Methods. (D) MHV titers in supernatants were determined as previously described (52). Two or three replicates were performed in each experiment, and one of three independent experiments is shown.
FIG 2
FIG 2
MHV upregulates MDA5 and RIG-I expression to higher levels in BMM than in BMDC. (A) Basal mRNA levels of MDA5 and RIG-I in mock-infected BMM and BMDC were measured by real-time qPCR. Values that are significantly different (P < 0.05) from the value for BMM are indicated by an asterisk. (B and C) mRNA levels of MDA5 and RIG-I in BMM (B) and in BMDC (C) at different times p.i. were measured by real-time qPCR. CT ratios to HPRT were calculated as described in Materials and Methods. MDA5 and RIG-I were upregulated to a greater extent in BMM than in BMDC (note the differences in scale). Two or three replicates were performed in each experiment, and one of three independent experiments is shown.
FIG 3
FIG 3
MHV-induced upregulation of IFN-β, MDA5, and RIG-I in BMM is dependent on IFN-α/β receptor (IFNAR). BMM isolated from WT, IFNAR−/−, or MyD88−/− mice were infected with MHV (A and C) or SenV (E) or transfected with poly(I ⋅ C) (D). (A) MHV titers were determined in cell supernatants at 16 hours postinfection as described in Materials and Methods. (B) mRNA was harvested from mock-infected BMM, and basal levels of IFN-β, MDA5, and RIG-I were measured by real-time qPCR. (C to E) mRNA was harvested 16 h after infection or transfection. mRNA levels were measured by real-time qPCR. CT ratios to HPRT are shown. MHV induced upregulation to a greater extent than poly(I ⋅ C) or SenV did (note the differences in scale in panels B to E). Two or three replicates were performed in each experiment, and one of three independent experiments is shown. Values for IFNAR−/− or MyD88−/− BMM that were statistically significantly different from the values for wild-type cells are indicated as follows: *, P < 0.05; **, P < 0.01.
FIG 4
FIG 4
MHV infection results in greater cytokine and chemokine upregulation in BMM than in BMDC. BMM and BMDC were infected with MHV. (A) mRNA levels of CXCL10, CCL2, TNF, and IL-12 p40 were measured by real-time qPCR in BMM and BMDC at the indicated times p.i. ND, not detectable. (B) Basal levels of CXCL10, CCL2, and TNF were measured by real-time qPCR in mock-infected BMM and BMDC. CT ratios to HPRT are shown in panels A and B (note the differences in scale). In panels A and B, three or four replicates were performed in each experiment, and one of three independent experiments is shown. (C) Protein levels of TNF and IL-12 p70 in BMM were measured by ELISA at different times p.i. (D) Protein levels of TNF and CXCL10 in BMM and BMDC were assayed at 24 h p.i. In panels C and D, one of two independent experiments is shown. Values for BMDC that are significantly different from the value for BMM are indicated as follows: *, P < 0.05; **, P < 0.01.
FIG 5
FIG 5
MHV induces IFNAR-dependent and MyD88-dependent upregulation of cytokines and chemokines. (A) BMM isolated from WT, IFNAR−/−, or MyD88−/− mice were mock infected, infected with MHV or SenV, or transfected with poly(I ⋅ C) for 16 h. mRNA levels of CXCL10 and TNF were measured by real-time qPCR. (B) mRNA levels of IL-10, ISG15, IL-6, and IL-12 p40 in mock-infected or MHV-infected BMM isolated from WT, IFNAR−/−, or MyD88−/− mice were measured by real-time qPCR at 16 h p.i. ND, not detectable. CT ratios to HPRT are shown in panels A and B (note the differences in scale). (C) Protein levels of CXCL10, TNF, and IL-12 p70 were measured by ELISA. Two or three replicates were performed in each experiment, and one of two independent experiments (for panel C) or one of three independent experiments (for panels A and B) is shown. Values that are statistically significantly different from the value for infected WT BMM are indicated as follows: *, P < 0.05; **, P < 0.01.
FIG 6
FIG 6
Exogenously added IFN is required for maximal levels of cytokine and chemokine expression in BMM at 8 h p.i. and in BMDC and fibroblasts at 16 h p.i. BMM, BMDC, and 17Cl-1 cells were infected with MHV. Some samples were treated with 800 U of IFN-β at 4 h p.i. (A) Supernatants were harvested at 16 h p.i., and virus titers were measured. (B to E) RNA was prepared from mock-infected or MHV-infected BMM, BMDC, or 17Cl-1 cells at 8 or 16 h p.i. mRNA levels of type I IFN (IFN-β and IFN-α4) (B), RNA helicases (MDA5 and RIG-I) (C), IFN-dependent genes (CXCL10 and ISG15) (D), and an IFN-independent gene (TNF) (E) were measured by real-time qPCR. To determine whether upregulation required active virus replication, BMM were exposed to UV-inactivated virus prior to IFN treatment, and the levels of select genes were determined (shown in the boxes to the right of the graph for BMM 16-h sample graphs). CT ratios to HPRT are shown. All genes were induced to higher levels in BMM than in BMDC or 17Cl-1 cells (note the differences in scale). Three or four replicates were performed in each experiment, and one of three independent experiments is shown. Values that were statistically significantly different from the values for cells infected with MHV and treated with IFN are indicated as follows: *, P < 0.05; **, P < 0.01.
FIG 7
FIG 7
Exogenously added IFN enhances expression of CXCL10 but not TNF. BMM, BMDC, and 17Cl-1 cells were infected with MHV with (+) or without (−) IFN-β treatment at 4 h p.i. The levels of CXCL10 (A) and TNF (B) protein were determined by an ELISA. One of two independent experiments is shown. Values that were significantly different from the values for MHV-infected, IFN-treated cells (P < 0.01) are indicated (**).
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