ALV-J strain SCAU-HN06 induces innate immune responses in chicken primary monocyte-derived macrophages

Abstract

Avian leucosis virus subgroup J (ALV-J) can cause lifelong infection and can escape from the host immune defenses in chickens. Since macrophages act as the important defense line against invading pathogens in host innate immunity, we investigated the function and innate immune responses of chicken primary monocyte-derived macrophages (MDM) after ALV-J infection in this study. Our results indicated that ALV-J was stably maintained in MDM cells but that the viral growth rate was significantly lower than that in DF-1 cells. We also found that ALV-J infection significantly increased nitric oxide (NO) production, but had no effect on MDM phagocytic capacity. Interestingly, infection with ALV-J rapidly promoted the expression levels of Myxovirus resistance 1 (Mx) (3 h, 6 h), ISG12 (6 h), and interleukin-1β (IL-1β) (3 h, 12 h) at an early infection stage, whereas it sharply decreased the expression of Mx (24 h, 36 h), ISG12 (36 h), and made little change on IL-1β (24 h, 36 h) production at a late infection stage in MDM cells. Moreover, the protein levels of interferon-β (IFN-β) and interleukin-6 (IL-6) had sharply increased in infected MDM cells from 3 to 36 h post infection (hpi) of ALV-J. And, the protein level of interleukin-10 (IL-10) was dramatically decreased at 36 hpi in MDM cells infected with ALV-J. These results demonstrate that ALV-J can induce host innate immune responses and we hypothesize that macrophages play an important role in host innate immune attack and ALV-J immune escape.

Keywords: ALV-J; innate immune response; macrophage.

Figure 1.

Morphological changes of chicken MDM. Chicken MDM cells at 6 h, 2 d, 4 d, and 6 d post-adherence as viewed using an inverted microscope (a, b, c, d) (300×). Morphological comparisons between normal MDM (e, 8,000×) and ALV-J infected MDM (f, 10,000×) using scanning electron microscopy.

Figure 2.

Expression of surface molecules on chicken MDM cells. Cultured MDM were stained with monoclonal antibody KUL01-FITC and MHC-II and analyzed using flow cytometry.

Figure 3.

Growth kinetics of SCAU-HN06 propagated in DF1 and MDM cells. Growth curves for SCAU-HN06 in (a) DF1 and (b) MDM cells by measuring expression of the viral group-specific antigen p27 by ELISA from cell culture supernatants collected at 3 to 168 hpi. (c) PCR results using primers specific for SCAU-HN06 in infected MDM and uninfected MDM at 3 to 36 h.

Figure 4.

Detection of NO and phagocytic capacity in MDM after SCAU-HN06 infection. (a) NO measurements using a commercial kit (b) Flow cytometry of FITC-dextran loaded normal MDM and infected MDM at 24 hpi. (c) Median fluorescence intensity (MFI) values between control and infected groups (*P < 0.05, ns P > 0.05).

Figure 5.

Relative quantification of mRNA expression of Mx and ISG12 in MDM infected with SCAU-HN06. (a) Mx; (b) ISG12 (**P < 0.01; ***P < 0.001).

Figure 6.

Cytokine production in MDM cells at 3 to 36 hpi. Cytokines were measured by ELISA. (a) IL-1β; (b) IL-6; (c) IL-10; (d) IFN-β (**P < 0.01; ***P < 0.001).