TNF-alpha, produced by feline infectious peritonitis virus (FIPV)-infected macrophages, upregulates expression of type II FIPV receptor feline aminopeptidase N in feline macrophages

Abstract

The pathogenicity of feline infectious peritonitis virus (FIPV) is known to depend on macrophage tropism, and this macrophage infection is enhanced by mediation via anti-S antibody (antibody-dependent enhancement, ADE). In this study, we found that TNF-alpha production was increased with viral replication in macrophages inoculated with a mixture of FIPV and anti-S antibody, and demonstrated that this culture supernatant had feline PBMC apoptosis-inducing activity. We also demonstrated that the expression level of the FIPV virus receptor, feline aminopeptidase N (fAPN), was increased in macrophages of FIP cats. For upregulation of TNF-alpha and fAPN in macrophages, viral replication in macrophages is necessary, and their expressions were increased by ADE of FIPV infection. It was demonstrated that a heat-resistant fAPN-inducing factor was present in the culture supernatant of FIPV-infected macrophages, and this factor was TNF-alpha: fAPN expression was upregulated in recombinant feline TNF-alpha-treated macrophages, and FIPV infectivity was increased in these macrophages. These findings suggested that FIPV replication in macrophages increases TNF-alpha production in macrophages, and the produced TNF-alpha acts and upregulates fAPN expression, increasing FIPV sensitivity.

Fig. 1

Relationship between FIP infection and TNF-alpha production in feline macrophages. SPF cat-derived macrophages (2 × 10⁶ cells) were cultured with medium alone, FIPV, FIPV + α-FIPV S MAb, and α-FIPV MAb alone for 3 days, and the cells and culture supernatant were collected. The virus titer in the culture supernatant was measured by plaque assay (A, left), and TNF-alpha production was measured using cytotoxicity against WEHI-164 cells as an index (A, right). In addition, the FCoV N gene (B, left) and TNF-alpha mRNA (B, right) in the cells were detected by RT–PCR. The FCoV N gene and TNF-alpha mRNA were quantitatively analyzed in terms of the relative density value to the mRNA for the housekeeping gene GAPDH. n = 5, **p < 0.01. N.D.: not detected.

Fig. 2

TNF-alpha production in FIPV-infected macrophages depends on viral replication. SPF cat-derived macrophages (2 × 10⁶ cells) were inoculated with a mixture of heat-inactivated FIPV and α-FIPV S MAb (white circle) or a mixture of live FIPV and α-FIPV S MAb (black circle), and the cells and culture supernatant were collected daily for 3 days. (A) TNF-alpha mRNA in the cells was detected by RT–PCR. TNF-alpha mRNA was quantitatively analyzed in terms of the relative density value to the mRNA for the housekeeping gene GAPDH. (B) TNF-alpha production in the culture supernatant was measured using cytotoxicity against WEHI-164 cells as an index. n = 5, **p < 0.01.

Fig. 3

PBMC apoptosis-inducing activity of FIV-infected macrophage culture supernatant. SPF cat-derived macrophages (2 × 10⁶ cells) were cultured with medium alone, FIPV, FIPV + α-FIPV S MAb, and α-FIPV MAb alone, and the culture supernatants were collected after 3 days. The PBMC of SPF cats (2 × 10⁶) were cultured at 37 °C for 4 h in the presence of each culture supernatant, and apoptotic cells were detected by TUNEL. n = 6, **p < 0.01.

Fig. 4

FCoV N gene, TNF-alpha, and fAPN mRNA expression levels in macrophages of FIP and SPF cats. Alveolar macrophages (2 × 10⁶ cells) were collected from FIP and SPF cats, and the FCoV N gene (A), TNF-alpha mRNA (B), and fAPN mRNA (C) were detected by RT–PCR. The FCoV N gene, TNF-alpha mRNA, and fAPN mRNA were quantitatively analyzed in terms of the relative density value to the mRNA for the housekeeping gene GAPDH. n = 7, N.D.: not detected.

Fig. 5

Relationship between fAPN mRNA expression and FIPV infection in cultured feline macrophages. SPF cat-derived macrophages (2 × 10⁶ cells) were cultured with medium alone, FIPV, FIPV + α-FIPV S MAb, and α-FIPV MAb alone. The cells were collected after 3 days, and the FCoV N gene and fAPN mRNA were detected by RT–PCR. FCoV N gene and fAPN mRNA were quantitatively analyzed in terms of the relative density value to the mRNA for the housekeeping gene GAPDH. n = 5, ^**p < 0.01. N.D.: not detected.

Fig. 6

fAPN expression in feline macrophages treated with FIPV-infected macrophage culture supernatant. SPF cat-derived macrophages (2 × 10⁶ cells) were inoculated with a mixture of FIPV and α-FIPV S MAb. The culture supernatant was collected after 3 days, and heated at 56 °C for 30 min to inactivate viruses. SPF cat-derived macrophages (2 × 10⁶ cells) were cultured with medium containing 10% inactivated culture supernatant. The cells were collected on days 0, 1, 3, and 5 of culture, and the intracellular fAPN mRNA (A) and cell surface fAPN (B) expression levels were measured. fAPN mRNA was quantitatively analyzed in terms of the relative density value to the mRNA for the housekeeping gene GAPDH. White circle: Treatment with control macrophage culture supernatant, black circle: treatment with culture supernatant of macrophages inoculated with a mixture of FIPV and α-FIPV S MAb. n = 5, **p < 0.01 vs. treatment with culture supernatant of intact (control) macrophages.

Fig. 7

Changes in fAPN expression level in macrophages treated with recombinant feline TNF-alpha. SPF cat-derived macrophages (2 × 10⁶ cells) were cultured with medium containing 1 and 10 ng/ml recombinant feline TNF-alpha. The cells were collected on days 0, 1, 3, and 5 of culture, and the intracellular fAPN mRNA (A) and cell surface fAPN (B) expression levels were measured. fAPN mRNA was quantitatively analyzed in terms of the relative density value to the mRNA for the housekeeping gene GAPDH. n = 5, **p < 0.01 vs. TNF-alpha 0 ng/ml.

Fig. 8

FIPV replication in macrophages treated with recombinant feline TNF-alpha. SPF cat-derived macrophages (2 × 10⁶ cells) were cultured with medium containing 1 and 10 ng/ml recombinant feline TNF-alpha for 5 days. The cells were then inoculated with FIPV, and the culture supernatants were collected every 12 h for 2 days. The virus titers in the supernatants were measured by plaque assay. White bar: without TNF-alpha, gray bar: 1 ng/ml TNF-alpha was added, black bar: 10 ng/ml TNF-alpha was added. n = 4, **p < 0.01.