Molecular characterization of mudskipper (Boleophthalmus pectinirostris) hypoxia-inducible factor-1α (HIF-1α) and analysis of its function in monocytes/macrophages

Abstract

Hypoxia-inducible factor-1α (HIF-1α) plays a critical role in immune and inflammatory responses and is important in controlling a variety of processes in monocytes and macrophages. However, very little information is available about the functions of HIF-1α in fish monocytes/macrophages (MO/MФ). In this study, the cDNA sequence of the mudskipper (Boleophthalmus pectinirostris) HIF-1α gene (BpHIF-1α) was determined. Sequence comparison and phylogenetic tree analysis showed that BpHIF-1α is clustered in the fish HIF-1α tree. Constitutive expression of BpHIF-1α mRNA was detected by real-time quantitative PCR in all tested tissues, and the expression was found to be dramatically increased in the skin, liver, spleen, and kidney after Edwardsiella tarda infection. In addition, hypoxia and infection induced the expression of the BpHIF-1α transcript and protein in MO/MФ, respectively. Hypoxia caused an increase in phagocytic and bactericidal capacity of mudskipper MO/MФ in a BpHIF-1α-dependent manner. BpHIF-1α induced an anti-inflammatory status in MO/MФ upon E. tarda infection and hypoxia. Therefore, BpHIF-1α may play a predominant role in the modulation of mudskipper MO/MФ function in the innate immune system.

Fig 1. Alignment of the deduced amino acid sequence of mudskipper HIF-1α (Bold) with the same protein from other fishes.

The alignment was generated using BioEdit version 7.2. Dashes indicate gaps introduced to facilitate alignment. The bHLH domain, PAS-A and PAS-B domains, ODD domain, and TAD-N and TAD-C domains are indicated.

Fig 2. Phylogenetic tree comparing the sequence of mudskipper HIF-1α (Bold) with that of other vertebrate species.

The scale bar refers to evolutionary distances in substitutions per site. The numbers at tree nodes refer to percentage bootstrap values after 1,000 replicates.

Fig 3. RT-qPCR analysis of HIF-1α mRNA expression in various mudskipper tissues.

Fish were euthanized at 3, 6, 12, and 24 h after intraperitoneal injection of E. tarda, and PBS was used as the control. L, liver; Sp, spleen; K, kidney; H, heart; G, gill; I, intestine; Sk, skin. HIF-1α transcript levels were normalized to those of 18S rRNA. Data are expressed as mean ± SEM (n = 4 for tissues). *, P< 0.05.

Fig 4. RT-qPCR analysis of mRNA expression in the skin and intestine after E. tarda infection.

Fish were euthanized at 3, 6, 12, and 24 h after intraperitoneal injection of E. tarda, and PBS was used as the control. Then, the skin (A and B) and intestine (C and D) were collected. BpGlut1, BpVegf-a, BpAldoc, BpCSF1R, and BpiNOS transcript levels were normalized to those of 18S rRNA. Data are expressed as the mean ± SEM (n = 4 for tissues). *, P< 0.05.

Fig 5. HIF-1α expression in MO/MΦ of mudskippers after infection with E. tarda.

(A) Flow cytometry analysis of the percentage of CSF1R-positive cells in mudskipper kidney-derived MO/MΦ following incubation with anti-CSF1R IgG or isotype IgG. Histogram represents the percentage of CSF1R-positive cells in the anti-CSF1R IgG or isotype IgG-treated groups. (B and D) MO/ MΦ were collected at 3, 6, 12, and 24 hpi with E. tarda, and PBS was used as the control. The transcript levels of HIF-1α and HIF-1α target genes were normalized to those of 18S rRNA. Data are expressed as mean ± SEM for four independent experiments. *, P < 0.05. (C) Western blot analysis of mature BpHIF-1α protein in MO/MФ at 0, 3, 6, 12, and 24 hpi with E. tarda. Histogram displays the changes in relative band intensity of BpHIF-1α protein after infection. Quantification of BpHIF-1α protein expression is presented as fold change over β-actin, which was assigned a unit of 100. Data are representative of three independent experiments. *, P < 0.05.

Fig 6. Alteration of HIF-1α expression in MO/MΦ of mudskippers cultured in parallel with either normoxia or hypoxia.

(A and C) MO/ MΦ were collected at 3, 6, 12, and 24 h post normoxia or hypoxia, incubated in normoxia (21% O₂) or in hypoxia (1% O₂). The transcript levels of HIF-1α and HIF-1α target genes were normalized to those of 18S rRNA. Data are expressed as mean ± SEM for four independent experiments. *, P < 0.05. (B) Western blot analysis of mature BpHIF-1α protein in MO/MФ at 0, 3, 6, 12, and 24 h post hypoxia. Histogram displays the changes in relative band intensity of BpHIF-1α protein on hypoxia. Quantification of BpHIF-1α protein expression is presented as fold change over β-actin, which was assigned a unit of 100. Data are representative of three independent experiments. *, P < 0.05.

Fig 7. Hypoxia-influenced phagocytosis of mudskipper MO/MΦ.

(A) Histogram displays the effect of BpHIF-1α siRNA transfection on MO/MΦ BpHIF-1α mRNA expression in a hypoxic environment, as detected by RT-qPCR. Transcript levels of BpHIF-1α were normalized to those of 18S rRNA. Data are expressed as mean ± SEM for four independent experiments. *, P < 0.05. (B) MO/MΦ were transfected with 100 pmol BpHIF-1α siRNA (H siRNA) or 100 pmol scrambled siRNA (sc siRNA) in 250 μl of Opti-MEM for the indicated length of time under normoxic (N, −) or hypoxic (H, +) conditions, respectively. Then, FITC–DH5α was added at an MOI of 20, followed by incubation for 1 h. The phagocytosed bacteria were investigated by flow cytometry assays. Quantification of phagocytosis was presented as fold change over the control, which was assigned a unit of 100. Data are expressed as mean ± SEM for three separate experiments with a total of 100,000 cells per experiment. *, P< 0.05.

Fig 8. Effects of hypoxia on MO/MΦ bactericidal activity.

Hypoxic MO/MΦ retained the ability to eradicate E. tarda. (A) Histogram displays the effect of BpHIF-1α siRNA transfection on MO/MΦ BpHIF-1α mRNA expression after infection, as detected by RT-qPCR. Transcript levels of BpHIF-1α were normalized to those of 18S rRNA. Data are expressed as mean ± SEM for four independent experiments. *, P < 0.05. (B) Standard curve was generated from 10-fold serial dilutions of E. tarda, as detected by RT-qPCR. (C) MO/MФ were transfected with scrambled siRNA or BpHIF-1α siRNA for the indicated length of time. E. tarda bacterial cells were added at an MOI of 10 and incubated for additional 30 min. Viable bacterial cells were enumerated by RT-qPCR, as described in Materials and Methods. Histograms represent the viability rate (%) of bacteria. Each bar represents mean ± SEM of the results from four independent experiments. *, P < 0.05.

Fig 9. Effects of hypoxia on the expression of cytokines in mudskipper MO/MΦ challenged with viable E. tarda cells.

MO/MΦ were incubated under normoxic or hypoxic conditions. E. tarda cells were added to the culture media at an MOI of 10. Normoxia-treated MO/MΦ were used as the control. (A) MO/MΦ were subjected to normoxia or hypoxia for 3, 6, 12, and 24 h. At each time point, BpIL-1β, BpTNFα, BpIL-10, or BpTGFβ transcript is presented as the fold change over the control, which was normalized to Bp18S rRNA using the 2^−ΔΔCt method. Data are expressed as mean ± SEM for four independent experiments. *, P < 0.05. (B) BpIL-10 protein expression was measured after E. tarda infection and hypoxia in MO/MΦ by ELISA. Quantification of BpIL-10 protein expression was presented as fold change over the value at 3 h, which was assigned a unit of 1. Data are expressed as mean ± SEM of the results from four independent experiments. *P < 0.05. (C) MO/MΦ were incubated with scrambled siRNA (sc siRNA) or BpHIF-1α siRNA (H siRNA) at 12 h post normoxia or hypoxia, respectively. Normoxia-treated MO/MΦ were used as the control. The cytokine transcript is presented as fold change over the control, which was normalized to Bp18S rRNA using the 2^−ΔΔCt method. Data are expressed as mean ± SEM for four independent experiments. *, P < 0.05.