IFN1 Enhances Thrombocyte Phagocytosis through IFN Receptor Complex-JAK/STAT-Complement C3.3-CR1 Pathway and Facilitates Antibacterial Immune Regulation in Teleost

IFNs belong to class II α-helical cytokines along with the IL-10 family, involved in innate and adaptive immunity in jawed vertebrates (1). Currently, IFNs are classified into four types: types I, II, III, and IV IFNs (2). Types I, III, and IV IFNs are the primary antiviral cytokine lineages, whereas type II IFN mainly contributes to protection against bacterial, fungal, and parasitic pathogens (3). Over half of vertebrates are teleosts, which are economically and evolutionarily important. Teleosts contain types I, II, and IV IFNs, but not type III IFN (1, 4), of which type I IFNs are highly diverse and are divided into three groups (I–III). Group I IFNs exist in all the teleost species, whereas group II IFNs appear to be limited to certain species/lineages, such as salmonids, cyprinids, and some perciformes, and group III are found, to date, only in salmonids within teleosts (5). Group I IFNs are composed of four subgroups (IFNa, d, e, h), group II comprise two subgroups (IFNb, c), and group III contains only one subgroup (IFNf) (5). Every subgroup contains one to several members. A model animal zebrafish (Danio rerio) has four type I IFN members: IFN-φ1 (IFNa), IFN-φ2 (IFNc), IFN-φ3 (IFNc), and IFN-φ4 (IFNd) (6, 7). For the classic type I IFN antiviral function, viruses invade the host and induce type I IFN via pattern recognition receptor (PRR) signaling pathways, then type I IFN facilitates IFN-stimulated genes (ISGs) for antiviral effectors via the type I IFN receptor (IFNAR1 and IFNAR2)-JAK/STAT pathway (8, 9). Correspondingly, type I IFN receptors in teleosts are cytokine receptor family B1 (CRFB1) and CRFB5 for group I IFNs and CRFB2 and CRFB5 for group II IFNs, respectively (6). However, the latest study found that grass carp IFN1 interacts with CRFB1, CRFB2, and CRFB5 receptors simultaneously, and the antiviral signal is subsequently delivered through the JAK/STAT pathway (10). In addition to inducing an antiviral immune response, type I IFN also exhibits complex functions in host defense against bacterial infections. When the host is infected with Listeria monocytogenes or Mycobacterium tuberculosis, high concentrations of type I IFNs are harmful to the host because they prevent B cell responses, which leads to the production of immunosuppressive molecules and reduces the responsiveness of macrophages to IFN-γ activation (11, 12). Zebrafish IFN-φ1 protects the host from Streptococcus iniae (Gram-positive bacteria) infection (13), and Mus musculus IFN-β (MmIFN-β) can directly kill multiple strains of Gram-positive bacteria but is less effective against the Gram-negative strains of bacteria in vitro (14). A recent study found that some type I IFNs with strong positive charges possess robust antibacterial activity in vitro in nonmammalian jawed vertebrates (15). However, the antibacterial mechanisms in vivo remain unknown.

Cytokines exert immune-regulatory functions via immune cells. Blood, consisting of erythrocytes, thrombocytes, and leukocytes, plays a crucial role in immune defense. Leukocytes are major immune cells in blood. Thrombocytes also play an important role in immune defense, and they are the second most abundant blood cells, outnumbered only by erythrocytes (16). Thrombocytes are the crucial components in vertebrate blood, with clotting activity and other immune functions. Thrombocytes in nonmammalian vertebrates have a cell nucleus and can express more than 300 proteins, including proinflammatory cytokines, chemokines, cell adhesion proteins, etc., showing their potential to participate in the process of antimicrobial infections (17). Meanwhile, TLRs expressed by thrombocytes sense the microbe-associated molecular patterns of bacteria and viruses to activate immune responses (18–20). In addition to immune regulation, phagocytosis is also a crucial immune function of thrombocytes. Common carp (Cyprinus carpio) thrombocytes can directly ingest fluorescent latex microspheres or bacteria with a diameter of 0.5–3 μm, form phagolysosomes, and facilitate antibacterial effect. This phagocytosis is even stronger under some conditions other than that of B cells. Moreover, the phagocytosis can also be enhanced by serum opsonization (21). Further study finds that phagocytosis of common carp thrombocytes requires the activation factors secreted by other leukocytes (22). However, the mechanisms of thrombocyte phagocytosis and the regulatory signaling pathway are still unclear.

In the present study, grass carp (Ctenopharyngodon idella), an economical fish with the highest production in the world, was employed as the representative research species. Grass carp has four type I IFN members: IFN1 (IFNa), IFN2 (IFNc), IFN3 (IFNc), and IFN4 (IFNd) (6). The antibacterial mechanisms in vivo of grass carp IFN1 (corresponding to zebrafish IFN-φ1) were addressed by Ab blockade, specific inhibitor, cell screening by flow cytometry, Western blotting (WB) detection, etc. The results deepened the understanding of antibacterial mechanisms of type I IFNs in jawed vertebrates.

Grass carp for experiments were obtained from Luhu farm (Wuhan, China). The mice and rabbits (for Ab preparation) were obtained from the Laboratory Animal Center of Huazhong Agricultural University (Wuhan, China). All experiments were performed in accordance with the guidelines of the Laboratory Animal Center of Huazhong Agricultural University. The protocols were approved by the animal research ethics committee of Huazhong Agricultural University (HZAURAB-2021-0007).

Before the formal experiment, healthy grass carp weighing 25 ± 5 g were temporarily raised in a 300-L tank for at least 2 wk to maintain a stable state. The brain, hepatopancreas, trunk, kidney, blood, gill, intestine, spleen, skin, muscle, head kidney, heart, and eye tissues of healthy grass carp (n = 8) were collected and put into TRIzol (put into liquid nitrogen subsequently) for RNA extraction. mRNA expression was determined by quantitative real-time RT-PCR (qRT-PCR). 18S rRNA was employed as an internal reference (23). Primers for qRT-PCR analysis are listed in Supplemental Table II. The results were calculated by using the 2^−△△Ct method.

Aeromonas hydrophila (strain 1703) was isolated and preserved in our laboratory (15). A. hydrophila was grown overnight in Luria Bertani (LB) medium and reinoculated on the next day at 1:100 for 4 h. Bacteria in logarithmic growth phase were collected, centrifuged at 12,000 rpm for 5 min, and washed with PBS twice. A. hydrophila was suspended with PBS, each fish was i.p. injected with 1 × 10⁶ CFU bacteria (100 μl), and the control group was injected with the same volume of PBS. Head kidney, spleen, intestine, and blood were collected into TRIzol at 12 h, 24 h, 48 h, and 72 h after bacterial injection, respectively (n = 6). In addition, blood samples of grass carp at different time points after infection were treated with whole-cell lysates for WB detection. WB bands were analyzed on the basis of three independent replicates using ImageJ software.

The recombinant protein expression and purification of IFN1 and IFN3 (without signal peptide) were as previously described in our laboratory (15). For the expression and purification of MmIFN-β, His-IFN1, and His-CRFB2, the gene sequence was obtained from the National Center for Biotechnology Information for MmIFN-β (GenBank No. NM_010510.1), IFN1 (GenBank No. GU139255.1) and CRFB2 (GenBank No. KU193788.1). Primers were designed by Primer Premier 5 software to amplify MmIFN-β from mouse liver tissue and His-IFN1 and His-CRFB2 from the grass carp spleen cDNA template. The amplified product was then constructed into pET-28a plasmid, and transformed into Escherichia coli BL21 (DE3)-expressing strain to produce fusion protein. Buffer A1 (20 mM Tris-HCl, 1 M NaCl, 10% glycerol [pH 7.4]) was used to suspend the bacterial solution and purify the supernatant after high-pressure crushing. The nickel column was used for binding to His-tagged MmIFN-β, IFN1, and CRFB2, followed by sufficient washing with buffer A1 and buffer A2 (20 mM Tris-HCl, 500 mM NaCl, 10% glycerol [pH 7.4]) to remove miscellaneous protein, and finally buffer A3 (20 mM Tris-HCl, 10 mM imidazole, 200 mM NaCl, 10% glycerol [pH 7.4]) was used to elute MmIFN-β, IFN1, and CRFB2, and the recombinant MmIFN-β, IFN1, and CRFB2 protein were obtained by dialysis desalting and ultrafiltration.

mRNA sequences of CRFB1 (GenBank No. KF255603.1), CRFB2, CRFB5 (GenBank No. KF893262.1), and CR1 (GenBank No. ON714494) were obtained from the National Center for Biotechnology Information. Primers were designed to amplify CR1 and the extracellular region of CRBF1, CRBF2, and CRFB5 from the grass carp spleen cDNA template. After the amplified products were constructed into the pGEX-4T-1 plasmid, they were then transformed into E. coli BL21 (DE3) pLysS-expressing strain to express the recombinant protein with GST tag. The bacterial solution was suspended with buffer B1 (50 mM Tris-HCl, 5 mM EDTA [pH 8.0]), and the inclusion bodies were purified after high-pressure crushing. The inclusion bodies were washed three times by buffer B2 (50 mM Tris-HCl, 5 mM EDTA, 2 M urea [pH 8.0]) and buffer B3 (50 mM Tris-HCl, 5 mM EDTA, 2 M urea, 1% Triton [pH 8.0]), respectively, and then fully suspended with buffer B4 (0.1 M Tris-HCl, 10 mM DTT, 8 M urea [pH 8.0]) and subsequently shaken on a constant temperature shaker at 37°C for 1 h. The solution was centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was retained, and the recombinant protein was finally obtained by dialysis and religation, elution, and redialysis.

The purified IFN1, CRFB1, CRFB2, CRFB5, and CR1 (proteins were dialyzed into 20 mM Tris [pH 7.4] buffer) were s.c. injected into New Zealand rabbits with CFA (first time) and IFA (second and third times). One week after the third injection, the blood of the rabbits was taken, and the serum was gathered. The polyclonal Abs in rabbit antisera were purified by Ag-Ab affinity chromatography.

Leukocytes were isolated by Percoll density gradients as described previously (24). Erythrocytes were collected from the bottom layer of the Percoll separation medium that separates leukocytes. Thrombocytes were labeled with CD41 Ab (prepared previously in our laboratory) and obtained by flow cytometry (25).

The interactions of GST-CRFB1, 2, and 5 with His-IFN1 and of GST-CRFB1 and 5 with His-CRFB2 were determined by GST pull-down analysis according to a previously reported method (26). Briefly, 400 μl of GST-CRFB1, 2, and 5 or GST protein (control) was incubated with 40 μl GST fillers (Huiyan Bio, HQ030307100M) at room temperature for 1 h, and 400 μl of His-IFN1 was added to the mixture and incubated with gentle rocking for 2 h. After washing with TBS five times, the fillers were collected, and the samples were analyzed by WB with GST Ab (Abclonal, AE001) and His Ab (Abclonal, AE003). The interaction between GST-CRFB1 and 5 with His-CRFB2 refers to the above description.

In order to block the physiological IFN1 of grass carp in vivo, grass carp were i.p. injected with the purified IFN1 Abs or rabbit IgG at 10 μg/fish three times (Day [D] -3, D -1, and 0.5 h after challenge). To verify the receptors through which IFN1 regulates the phagocytosis of thrombocytes and the mechanism of IFN1 promoting phagocytosis, purified CRFB Abs (including CRFB1 Abs, CRFB2 Abs, and CRFB5 Abs) or CR1 Abs were used to block the receptors on the surface of thrombocytes, respectively. The experimental methods were described in a previous report (27). Peripheral blood thrombocytes were isolated by flow cytometry as per the above description. The cells were counted in a Neubauer chamber and adjusted to 1 × 10⁶ cells/ml and then incubated with rabbit IgG, GST Ab, CRFB1 Ab, CRFB2 Ab, CRFB5 Ab, or CR1 Ab (2 μg/ml) at 28°C for 6 h, respectively. After being washed three times with DMEM, the cells were collected and used in subsequent experiments.

The IFN1 blockade fish were infected with 1 × 10⁶ CFU bacteria (100 μl) A. hydrophila by i.p. injection. Pathological characteristics were monitored in one group, and the survival rate was counted (n = 30) for the next 7 d in another group. The blood, spleen, head kidney, and intestine (hindgut) tissues were collected on D1 and D3 after bacterial injection (n = 4). These tissues were homogenized, diluted with sterile PBS, and then spread on Rimler-Shotts medium plates (HB8584, Haibo). After culturing at 28°C for 16 h, A. hydrophila colonies on the plates were counted for tissue bacterial loads. The total RNAs were extracted from blood samples on D3 after infection, and mRNA expression levels of IL-1β, IL-2, MHC class IIα (MHC IIα), MHC IIβ, and complement 3.3 (C3.3) were detected by qRT-PCR. (Both MHC IIα and MHC Iiβ were examined. They show the same profiles, so we exhibit only MHC IIα in the Results section as a representative.) 18S rRNA was used as an internal gene. All primer sequences are shown in Supplemental Table I.

The leukocytes, erythrocytes, and thrombocytes were washed (cleaned with sterile PBS, with the lower layers collected after centrifugation at 500 × g for 15 min) three times and adjusted to 1 × 10⁶ cells/ml. Subsequently, the cells were stimulated by different concentrations of IFN1 (0, 0.5, 1, 1.5, 2 μg/ml) at 28°C in 5% CO₂ for 6 h. After the cells were washed three times with sterile PBS, 10 μl fluorescent microspheres (FITC-conjugated microspheres, 17155; Polysciences) or 1 × 10⁸ CFU/ml GFP-labeled E. coli (GFP-E. coli), together with 990 μl DMEM, were added to each tube (with cells) for the phagocytosis experiment. After incubating at 28°C in 5% CO₂ for 2 h, the cells that phagocytized GFP-E. coli were collected after washing three times with sterile PBS, and the cells that phagocytized fluorescent microspheres were isolated by gradient centrifugation. The phagocytosis rate was then analyzed by flow cytometry (MoFlo Astrios EQ; Beckman Coulter Life Sciences).

In order to observe phagocytosis of fluorescent microspheres and GFP-E. coli by thrombocytes, photographs were taken by immunofluorescence assay. The thrombocytes (1 × 10⁶ cells) were suspended with 50 μl PBS and evenly coated on the slide for air drying, and they were incubated with 4% paraformaldehyde for 15 min to fix the morphology, then washed with PBS three times, followed by incubation with DiI (Thermo Fisher; V2899) for 15 min, washing with PBS again three times, and finally incubation with DAPI (Thermo Fisher; 62248) for 10 min. After rinsing three times, treated slides were photographed by confocal microscopy (CFM; Nikon).

To investigate the localization of CR1 in thrombocytes, the cells (1 × 10⁶ cells) were suspended with 50 μl PBS and spread evenly on the glass slide. After 4% paraformaldehyde fixation and PBS washing, thrombocytes were incubated with 5% BSA at room temperature for 4 h and subsequently incubated with CR1 Ab (2 μg/ml) (GST Ab used as control) at 4°C for 12 h. After being washed three times, the cells were incubated with FITC-labeled fluorescent secondary Abs (Thermo Fisher; A16118) for 45 min, followed by staining with DAPI for 10 min, and finally filming with CFM.

To investigate whether thrombocytes kill bacteria after phagocytosis, and whether IFN1 can promote the bactericidal effect of thrombocytes after phagocytosis, cytochalasin D (MCE, HY-N6682) was employed to inhibit phagocytosis in thrombocytes. The thrombocytes (1 × 10⁶ cells) were stimulated with 1 μM cytochalasin D (dissolved in DMSO) or DMSO for 3 h, then they were stimulated with IFN1 (1 μg/ml, 6 h). Subsequently, the cells were incubated with A. hydrophila (1 × 10⁶ CFU/ml) for 4 h. After the cells were incubated with bacteria, the cells were cleaned with PBS containing ampicillin (10 min per incubation, three times) to remove the effects of extracellular bacteria. Medium (50 μl) was then taken to coat LB culture plates, and the number of colonies on the experimental groups of plates was counted after culturing at 37°C for 12 h.

Thrombocytes were stimulated with IFN1 (1 μg/ml) at 28°C for 6 h, and buffer-stimulated thrombocytes were used as a control. The treated thrombocytes were used for RNA extraction and subsequent sequencing. The expression level of each gene was calculated according to the transcripts per million reads method. RSEM (http://deweylab.biostat.wisc.edu/rsem/) was used to quantify gene abundance. Essentially, differential expression analysis was performed using the DESeq2/DEGseq/edgeR/Limma/NOIseq, differentially expressed genes (DEGs) with |log₂ (foldchange)| ≥ 1 and p-adjust ≤ 0.05 (DESeq2/edgeR/Limma)/p-adjust ≤ 0.001 (DEGseq)/Prob > 0.8 (NOIseq) were considered to be significant DEGs. In addition, functional enrichment analysis including GO (Gene Ontology; http://www.geneontology.org) and KEGG (Kyoto Encyclopedia of Genes and Genomes; http://www.genome.jp/kegg/) was performed to identify which DEGs were significantly enriched in GO terms and metabolic pathways at p-adjust ≤ 0.05 compared with the whole-transcriptome background. GO functional enrichment and KEGG pathway analysis were carried out by Goatools (https://github.com/tanghaibao/Goatools) and KOBAS (http://kobas.cbi.pku.edu.cn/home.do). The difference analysis results are listed in Supplemental Table II.

In order to verify IFN1 signaling via the JAK/STAT pathway in thrombocytes, thrombocytes were isolated and adjusted to 1 × 10⁶ cells. The cells were then stimulated with IFN1 (1 μg/ml) at 28°C for 30 min. STAT1 phosphorylation levels in thrombocytes were detected by STAT1 phosphorylation Ab (Abclonal, AP0109).

Fludarabine can effectively inhibit STAT1 signaling in humans (28). To study the inhibitory effect of fludarabine in grass carp cells, 5 μM fludarabine (diluted in DMSO buffer) (MCE; NSC118218) was incubated with Ctenopharyngodon idella kidney (CIK) cells at 28°C in 5% CO₂ for 24 h. Treated CIK cells were collected, RNA was extracted, and mRNA expression of downstream effectors myxovirus resistant (Mx) 1 and Mx3 was detected by qRT-PCR, using EF-1α as the internal gene (23). The results were calculated by using the 2^−△△Ct method.

To examine whether IFN1 signals through the JAK/STAT pathway to facilitate phagocytosis and activation of thrombocytes, the thrombocytes were isolated and adjusted to 1 × 10⁶ cells/ml and incubated with 5 μM fludarabine at 28°C in 5% CO₂ for 12 h. Pretreated cells were collected and washed three times with DMEM and then incubated with 1 μg/ml IFN1 for 4 h. One group of cells was washed again and then incubated with fluorescent microspheres or GFP-E. coli, and the phagocytosis rate was analyzed by flow cytometry subsequently. Another group of cells was collected, RNA was extracted, and expression levels of downstream genes IL-1β and MHC IIα and MHC IIβ were analyzed by qRT-PCR. (Both MHC IIα and MHC IIβ were examined. They show the same profiles, so we exhibit only MHC IIα in the Results section as a representative.) EF-1α was employed as the internal reference. The results were calculated by using the 2^−△△Ct method.

Each grass carp was i.p. injected with PBS, IFN3 (10 μg), or different doses (2.5, 5, 10, 20 μg) of IFN1, and the blood was collected from tail veins on D3 after injection. The blood samples were placed at room temperature for 1 h, followed by 4°C for 12 h to precipitate the sera. Furthermore, to identify whether the complement components in serum had a bactericidal effect, partial serum was treated in a 45°C air bath for 30 min to inactivate complement components. A. hydrophila and Streptococcus agalactiae (ATCC 13813) were employed as representative Gram-negative bacteria and Gram-positive bacteria, respectively. A total of 100 μl bacterial suspension at 1 × 10⁸ CFU/ml was incubated with 10 μl differently treated sera or PBS at room temperature for 1 h. The treated bacterial solution was plated on glass slides and dried, then incubated with DAPI (100 μg/ml) for 15 min. The bacterial aggregation was viewed by CFM. In addition, the aggregations of Yersinia ruckeri (DSM 18506) and Staphylococcus aureus (ATCC 25923) were detected.

In addition, differently treated serum or PBS were incubated with bacteria at room temperature for 3 h, and the treated bacterial solution was diluted and coated on LB culture plates. After 12-h culture, the colonies on the plates were counted, and the ratio between the experimental group and the PBS treatment group was denoted as antibacterial activity. Meanwhile, the bactericidal effects on A. hydrophila and S. agalactiae were also visually photographed and displayed.

The normal grass carp was i.p. injected with 1 × 10⁶ CFU A. hydrophila, and, 3 h later, each fish was injected with 10 μg IFN1, IFN3, or PBS. The pathological characteristics of some fish were observed every 6 h, and the deaths during the following 7 d were counted (n = 30). Besides, another group of fish blood, spleen, head kidney, and hindgut tissues were collected D1 and D3 after protein/PBS injection for the detection of tissue bacterial load (n = 4). Tissue wet/dry weight ratio was carried out as previously reported (29). In brief, the spleen, head kidney, and hepatopancreas of fish on D3 after infection and protein/PBS injection were collected, washed, and directly weighed as wet weight. After drying at 60°C for 2 d, the tissue was weighed and recorded as dry weight. The wet/dry weight ratio of each individual tissue was calculated (n = 6). Meanwhile, grass carp tissues including spleen, head kidney, and intestine (hindgut) were sampled on D3 after challenge, and they were immediately fixed with 10% neutral buffered formalin for 24 h and subsequently dehydrated, embedded in paraffin, and sectioned. Sectioned samples were mounted on aminopropyl triethoxysilane–coated slides. Following the deparaffinization in xylene, sections were rehydrated, stained with H&E, and mounted with neutral gum, then the images were captured.

In order to study the antibacterial immune responses to type I IFN injection in mice, LPS was used as a positive control because it stimulated the production of inflammatory cytokines and complements in mice (30). Each healthy mouse (6–8-wk-old female mice, 20 ± 2 g) was i.p. injected with 10 μg recombinant IFN-β, 10 μg LPS, or an equal volume of buffer (20 mM Tris-HCl [pH 7.4]). Blood samples were taken at 12, 24, 48, and 72 h after injection. An ELISA kit (Abcam; ab263886) was used to detect the content of C3 in serum. Meanwhile, 10 μl serum or PBS was incubated with 100 μl A. hydrophila or S. agalactiae (1 × 10⁸ CFU/ml) for 3 h. The treated solutions were diluted and coated on LB culture plates. After 12-h culture, the colonies on the plates were counted. Another part of blood samples was used for RNA extraction, and the expression of antiviral genes MmISG15 and MmMx1 was detected to verify the biological activity of MmIFN-β, and immune genes MmIL-1β, MmMHC IIα, and MmMHC IIβ were detected by qRT-PCR. (Both MmMHC IIα and MmMHC IIβ were examined. They show the same profiles, so we exhibit only MmMHC IIα in the Results section as a representative.) ACTB was employed as an internal gene (31). All primer sequences are shown in Supplemental Table I.

Data were presented as the mean ± SD of at least three replicates. Significant differences were analyzed using the Student t test for paired comparisons or one-way ANOVA for multiple comparisons. p < 0.05 was considered a statistically significant difference (*p < 0.05, **p < 0.01, and ***p < 0.001). The survival rate was calculated, and the survival curves are presented as Kaplan-Meier plots and statistically using a log-rank test. All statistical analyses were conducted using GraphPad 8.0 data view software.

A previous study revealed that grass carp IFN1 has a broad-spectrum robust direct bactericidal activity in vitro (15). We wondered whether this antibacterial activity is meaningful in vivo. First, we investigated the tissue distribution of IFN1 in healthy grass carp. mRNA transcripts showed that IFN1 is expressed, in all 12 tissues examined, with high expression in the brain and hepatopancreas (Fig. 1A). After challenge with A. hydrophila, IFN1 is distinctly induced in the major immune tissues. mRNA expression of IFN1 is significantly induced from 12 h after bacterial infection in head kidney (Fig. 1B) and spleen (Fig. 1C) and from 48 h in intestine (hindgut) (Fig. 1D). Notably, mRNA expression is significantly induced at 24 h in blood, and even more so at 48 h and 72 h (∼25-fold higher than that in control) (Fig. 1E). In order to further investigate IFN1 functions at the protein level in blood, we recombinantly expressed and purified intact grass carp IFN1 and subsequently prepared and purified rabbit polyclonal Ab to anti-IFN1 (Fig. 1F). Examined by WB, IFN1 protein is detectable in blood at 48 and 72 h after bacterial infection (Fig. 1G). These results indicated that bacterial infection can induce a large amount of IFN1 expression in grass carp tissues, especially in blood.

IFN1 was induced greatly in the blood of the bacteria-infected grass carp, so we wondered how IFN1 regulates hemocytes to exert antibacterial immune functions. First, we isolated erythrocytes, leukocytes, and thrombocytes from grass carp peripheral blood. Then, we tested the phagocytosis for fluorescent microspheres. Under normal conditions, all three types of blood cells have the ability to phagocytize fluorescent microspheres (Fig. 2A–2C, upper right corner). After 1 μg/ml IFN1 stimulation, all the phagocytosis rates rise, from 4.35% to 5.20% (increasing 19.54%) for erythrocytes (Fig. 2A), from 10.14% to 15.28% (increasing 50.69%) for leukocytes (Fig. 2B), and from 8.17% to 13.16% (increasing 70.30%) for thrombocytes (Fig. 2C), which indicated that IFN1 can facilitate the phagocytic activity of hemocytes and has the most obvious regulatory effect on thrombocytes. Furthermore, we tested the phagocytosis of thrombocytes to GFP-E. coli. The phagocytosis images of thrombocytes to fluorescent microspheres and GFP-E. coli were viewed and taken by CFM (Fig. 2D). We analyzed the phagocytosis rate of hemocytes to fluorescent microspheres after IFN1 stimulation, and the results showed that IFN1 has the most noticeable promoting effect on the phagocytosis of thrombocytes (Fig. 2E). Furthermore, we found the phagocytosis activities of thrombocytes for both fluorescent microspheres and GFP-E. coli are dependent on IFN1 concentration (Fig. 2F). Phagocytosis is closely associated with bacterial clearance. IFN1 promotes the killing of engulfed bacteria by thrombocytes (Fig. 2G). These results showed that IFN1 facilitates the phagocytosis and bactericidal effect of thrombocytes.

In addition, we explored the effect of IFN1 on the antibacterial activation of thrombocytes at the gene level. The analysis of thrombocytes stimulated by recombinant IFN1 were carried out by transcriptome sequencing and qRT-PCR verification. IFN1 significantly promotes mRNA expression of the inflammatory cytokine IL-1β (Fig. 3A) and the regulatory cytokine IL-2 (Fig. 3B). Meanwhile, the Ag presentation molecule MHC IIα (Fig. 3C) and the costimulatory signal CD80/CD86 (Fig. 3D), involved in Ag presentation and activation of T cells, are also significantly upregulated. Furthermore, we found that multiple complement components are obviously enriched in the IFN1-stimulated thrombocytes in transcriptome datasets (Supplemental Table II). Complement C3 activation and opsonization are critical parts of the host responses to infections in the extracellular environment (32). The complement system in grass carp contains nine C3 members (C3.1–C3.8 and C3-like) (33), and it is important to determine which member of C3 responds most to IFN1 stimulation. Therefore, we examined the expression profile of all the grass carp C3 members in IFN1-stimulated thrombocytes by qRT-PCR, and the results showed that IFN1 stimulation mainly induces the significant expression of C3.3 (Fig. 3E). Furthermore, mRNA expression of the complement receptor CR1 is also significantly increased after IFN1 stimulation (Fig. 3F). All the results indicated that IFN1 can promote the phagocytosis activity of blood cells, especially thrombocytes, and activate the immune-regulatory functions by inducing the expression of cytokines, Ag presentation–related molecules, and complement components, especially C3.3.

IFNs transmit antiviral signals through IFN receptor and JAK/STAT signaling (10, 11). To investigate whether IFN1 also transmits antibacterial immune signals through the same receptor (10) on thrombocytes, we examined the responses of these three subunits (CRFB1, CRFB2, and CRFB5) of receptors to bacterial or IFN1 stimulation in thrombocytes. The results showed that the expression levels of CRFB1, CRFB2, and CRFB5 are significantly upregulated (Fig. 4A–4C), which implies that IFN1 may transmit the antibacterial signals via CRFB1, CRFB2, and CRFB5. Furthermore, we verified the interaction between IFN1 with CRFB1, CRFB2, and CRFB5, as well as the interaction between CRFB2 with CRFB1 and CRFB5, through GST pull-down. The results showed that IFN1 can interact with CRFB1, CRFB2, and CRFB5 (Fig. 4D). CRFB2 can interact with CRFB1 and CRFB5 (Fig. 4E), whereas CRFB5 is generally considered to heterodimerize with CRFB1 or CRFB2 (34), which suggests the formation of a receptor complex between CRFB1, 2, and 5. Furthermore, IFN1 can promote STAT1 phosphorylation in thrombocytes (Fig. 4F). These results demonstrated that IFN1 signals through CRFB1/CRFB2/CRFB5-JAK/STAT in thrombocytes.

To obtain direct evidence that IFN1 signals through CRFB1/CRFB2/CRFB5 in antibacterial infection, we recombinantly expressed and purified the extracellular region of grass carp CRFB1, CRFB2, and CRFB5, and we prepared and purified rabbit polyclonal Abs (Supplemental Fig. 1A). Three Abs were used to block these receptor subunits on the thrombocyte membrane. The phagocytosis rate of the blocked thrombocytes to fluorescent microspheres and GFP-E. coli was detected by flow cytometry. The results showed that CRFB1, CRFB2, or CRFB5 Ab blockade abolishes the phagocytosis promoted by IFN1 in thrombocytes (Fig. 5A, 5B). Simultaneously, the blockade also reduces the expression of C3.3 in thrombocytes after IFN1 stimulation (Fig. 5C). These results indicated that IFN1 promotes phagocytosis and activation of thrombocytes via CRFB1/CRFB2/CRFB5. Subsequently, IFN1 signals through the JAK/STAT pathway in antiviral immunity (34, 35). We employed the STAT1 inhibitor fludarabine to verify whether IFN1 delivers prophagocytosis and activation signals through the JAK/STAT pathway. First, fludarabine reduces the promoting effect of IFN1 on the expression of Mx1 and Mx3 in CIK cells (Supplemental Fig. 1B, 1C), which confirmed the effectiveness of fludarabine in grass carp cells. Subsequently, the thrombocytes were pretreated with fludarabine and used for a phagocytosis experiment after IFN1 stimulation. The results demonstrated that the phagocytosis facilitated by IFN1 to GFP-E. coli and fluorescent microspheres is alleviated by fludarabine (Fig. 5D). Meanwhile, mRNA expression of IL-1β and MHC IIα induced by IFN1 stimulation is also reduced by fludarabine (Fig. 5E). In addition, pretreatment with fludarabine greatly impairs the bactericidal effect of thrombocytes activated by IFN1 (Fig. 5H).

The opsonic phagocytosis involving complement is an important type of phagocytosis (36). Thrombocytes express multiple complement system members and may exert complement-mediated opsonic phagocytosis (19). Therefore, we investigated in detail whether the phagocytosis of thrombocytes promoted by IFN1 relates to the complement system. First, we recombinantly expressed and purified the CR1 protein, followed by prepared and purified CR1 Ab (Supplemental Fig. 1D). Then, the immunofluorescence images showed that CR1 localizes on the membrane of thrombocytes (Fig. 6A). After that, IFN1 promotes the expression of CR1 of thrombocytes, whereas fludarabine inhibits this promotion effect (Fig. 6B). Subsequently, we blocked CR1 of thrombocytes for a phagocytosis assay. The results showed that the blockade of CR1 almost abolishes the prophagocytosis of IFN1 (Fig. 6C, 6D). Moreover, after blocking CR1, the bactericidal effect enhanced by IFN1 in thrombocytes is also significantly weakened (Fig. 6E). These results suggested that IFN1 enhances phagocytosis through the CRFB1/CRFB2/CRFB5-JAK/STAT-C3.3-CR1 pathway and antibacterial activation through the IFN receptor complex-JAK/STAT axis.

The complement system plays a crucial role in the antibacterial immune response in serum. First, we injected grass carp with recombinant IFN1 and collected the serum on D3. Then, we investigated the antibacterial activity (inactivated serum complements at 45°C for 30 min) of serum, which reflected the production of complements. The results indicated that IFN1 can facilitate the production of complements in serum in a dose-dependent manner (Fig. 7A–7C). Meanwhile, treatment at 45°C for 30 min cannot reduce the bactericidal activity of IFN1 itself (Supplemental Fig. 2A, 2A′), which indicated that the enhanced bactericidal activity is caused not by IFN1, but by the production of more complements. Subsequently, we examined mRNA expression of C3.3 and CR1 in blood after recombinant IFN1 injection, with recombinant IFN3 (a negatively charged type I IFN member without antimicrobial activity in vitro [15]) as a control (Fig. 7D). Grass carp blood continuously highly expresses C3.3 (Fig. 7E) and CR1 (Fig. 7F) from 24 h after IFN1 injection. In addition, we wanted to examine the antibacterial activity visually by CFM. To our surprise, complements in serum demonstrate obvious bacterial aggregation activity, except for bactericidal activity, in response to both Gram-negative and Gram-positive bacteria (Fig. 7G, Supplemental Fig. 2B, 2C). The bactericidal and aggregation abilities are significantly reduced in serum after heat inactivation of complements (Fig. 7A, 7G). All these results suggested that IFN1 enhances the roduction of complements with bacteriolysis and bacterial aggregation in serum.

Because IFN1 shows effective defense against bacterial infection as above, we wondered whether the antibacterial activity has physiological significance. We performed IFN1 blockade in vivo and subsequent bacterial infection experiments in grass carp. Purified IFN1 Ab was injected three times to block IFN1 in grass carp, and grass carp was challenged with A. hydrophila (Fig. 8A). The survival rate of grass carp obviously decreased in the IFN1 blockade group (Fig. 8B). Correspondingly, the bacterial loads in blood, spleen, head kidney, and intestine tissues significantly increased in the IFN1 blockade group on D3 after challenge (Fig. 8C–8F). Meanwhile, IFN1 blockade inhibits the expression of key immune factors IL-1β (Fig. 8G), IL-2 (Fig. 8H), MHC IIα (Fig. 8I), and C3.3 (Fig. 8J) in blood on D3 after bacterial infection. These results indicated that IFN1 is involved in the antibacterial defense in vivo at physiological concentration. As further evidence, we carried out bacterial challenge and IFN1 rescue experiments in grass carp. Grass carp were injected with a semilethal dose of A. hydrophila, and IFN1, IFN3, or PBS was injected at 3 h after bacterial challenge. The survival rate increases in the IFN1 injection group (Fig. 9A). Correspondingly, the bacterial burdens in blood, spleen, head kidney, and intestine tissues are significantly reduced in the IFN1 injection group on D3 after challenge (Fig. 9B–9E). Moreover, the wet/dry weight ratios in spleen, head kidney, and hepatopancreas tissues were remarkably decreased in the IFN1 injection group on D3 after challenge, which means that edema caused by bacterial infection is suppressed by IFN1 (Fig. 9F–9H). In addition, the histopathological sections in the control group show more leukocyte infiltration and tissue vacuolization in spleen tissue (Fig. 9I), looser interrenal tissue and larger glomerular capsule in head kidney tissue (Fig. 9J), and more severe intestinal myolysis and more goblet cells in hindgut tissue (Fig. 9K), although the IFN1 injection grass carp exhibit relatively normal tissue sections. All the experiments confirmed that IFN1 possesses effective antibacterial activity in vivo.

Mammalian type I IFNs have limited direct antimicrobial activity, which contrasts with the potent direct bactericidal effect of type I IFNs in teleosts (15). However, the regulatory function of mammalian type I IFN in antibacterial immunity is unclear. To investigate whether antibacterial immune regulation of type I IFN is conserved, we selected MmIFN-β, an equivalent member (bactericidal activity in vitro) with grass carp IFN1, recombinantly expressed and purified (Fig. 10A) for verification. Every mouse was injected with MmIFN-β or LPS (positive control). The expression levels of downstream antiviral ISG15 and Mx1 mRNA were detected to evaluate the bioactivity of recombinant MmIFN-β. The results showed that both ISG15 and Mx1 were upregulated to varying degrees in the blood of mice injected with MmIFN-β (Supplemental Fig. 1E, 1F), demonstrating its bioactivity in vivo. An ELISA kit was used to detect the protein levels of complement C3 in the serum, and the results showed that C3 slightly increases after injection, but there was no significant difference from the control group (Fig. 10B). Furthermore, the bactericidal activity of MmIFN-β-stimulated mouse serum was not significantly different from that of the control group (Fig. 10C). Besides, mRNA expression of MmIL-1β and MmMHC IIα in blood cells of MmIFN-β injection mice showed almost no variation from the control mice (Fig. 10D, 10E). These results indicated that mouse type I IFN with weak direct bactericidal activity has no obvious effect on antibacterial immune regulation (Fig. 11).

Type I IFNs play an important role in the immune response against viral infection. During viral infection, type I IFNs induce an antiviral state in both virus-infected cells and uninfected cells by inducing gene transcription that interferes with multiple stages of the viral replication cycle through various mechanisms (37). By contrast, the functional performance of type I IFNs in response to bacterial infection is complicated; low concentrations of type I IFNs are required to induce cell-mediated immune responses, but high concentrations inhibit the activation of immune cells and are harmful to the host (11). Type I IFNs can be produced by almost every cell type, including leukocytes, fibroblasts, and endothelial cells (38). Although IFNs have long been known in mammals, it was not until 2003 that different research groups identified type I IFNs in zebrafish (D. rerio) (39), pufferfish (Tetraodon nigroviridis) (40) and Atlantic salmon (Salmo salar) (41). Different expression profiles reflect that IFNs play different roles in the anti-infection response. In rainbow trout (Oncorhynchus mykiss), type I IFNs are produced in a wide range of tissues and cells after polyinosinic:polycytidylic acid stimulation, whereas type II IFNs are mainly expressed in head kidney leukocytes (42), indicating that they function at the different sites. Our previous research revealed, to our knowledge, a novel function of grass carp IFN1, directly killing bacteria through membrane depolarization and destruction mechanisms, which is different from the traditional indirect regulation of antiviral function of type I IFNs through signaling pathways (15). This finding opens a door for lower vertebrate type I IFNs in antibacterial immunity. However, the antibacterial mechanism in vivo remains unknown.

In the present study, we found that IFN1 is significantly induced in the main immune tissues and blood in grass carp during bacterial infection, and even IFN1 protein can be detectable by WB in blood from 48 h after bacterial infection, which implies that IFN1 may play a crucial role in bacterial infection in blood. Blood undertakes the circulation of substances throughout the body; meanwhile, various blood cells can directly participate in innate and adaptive immune responses to protect the host (43). Blood cells are classified as erythrocytes, leukocytes, and thrombocytes in teleosts (44). The present study shows that IFN1 stimulation remarkably increases phagocytosis of all three types of blood cells, and thrombocytes have the highest increased phagocytosis rate (70.30%). Thrombocytes are the second most abundant cells in blood and are involved in bacterial and viral infections (25, 44). Thrombocyte phagocytosis requires stimulation by certain cytokines (22). Type I IFN (IFN I-3) can promote the phagocytosis of bacteria by B cells in Japanese flounder (Paralichthys olivaceus) (45). These findings point to the possibility that type I IFNs may activate phagocytosis of blood cells to resist bacterial infection. However, how type I IFNs regulate the phagocytosis of thrombocytes has been unclear until now.

IFN1 stimulation promotes significant expression of IL-1β, IL-2, MHC II, and CD80/CD86 in thrombocytes. IL-1β is a classic early inflammatory factor, and high levels of IL-1β can rapidly stimulate immune cells to produce an immune response at the inflammatory site. IL-2 is an important cytokine that regulates the immune response and fights infection, and it is also an indispensable stimulus signal for activating T cells in the process of Ag presentation. MHC II is an important Ag presentation molecule, and CD80/CD86 is a costimulatory signaling molecule in Ag presentation. These suggest that IFN1 promotes the phagocytosis of bacteria by thrombocytes, killing bacteria intracellularly and possibly presenting Ags to T cells to activate adaptive immunity. Meanwhile, regulatory cytokines (IL-1β and IL-2) are secreted to further activate T cells. Human platelets have been reported to be involved in adaptive immunity (46), and avian thrombocytes also express adaptive immune-related molecules (47). Here, the antibacterial functions of piscine thrombocytes linking innate and adaptive immunity are activated by IFN1.

Type I IFNs bind to the heterodimeric receptor to activate immune responses in mammals. The receptor complex consists of IFNAR1 and IFNAR2, both of which belong to the class II cytokine receptor family (48). Grass carp IFN1 has been reported to bind to three subunit receptors, CRFB1, CRFB2, and CRFB5, in antiviral immunity (10). Type I IFNs bind to their receptor and promote the phosphorylation of STAT to activate the transcription of downstream ISGs in virus-induced responses (34). In grass carp, both JAK1 and TYK2 are upregulated and phosphorylated upon stimulation with polyinosinic:polycytidylic acid, and the overexpression of the type I IFN receptor chains CRFB1 and CRFB5 also promote JAK1 and TYK2 phosphorylation (49). The JAK/STAT pathway is a major signal transducer for a variety of cytokines and growth factors, affecting cell proliferation, differentiation, migration, apoptosis, etc. (50). However, whether this signal regulation axis (IFN-receptor-JAK/STAT) is involved in the immune response to bacterial infections remains unclear. We investigated the expression levels of CRFB1, CRFB2, and CRFB5 in thrombocytes under bacterial infection or IFN1 stimulation and found that they can be significantly upregulated. Furthermore, we confirmed the relationship between CRFB1, CRFB2, and CRFB5 to form a receptor complex, and we proved the direct interaction between IFN1 with CRFB1, CRFB2, and CRFB5. Also, IFN1 promotes STAT1 phosphorylation in thrombocytes. By Ab blockade of IFN1 receptor and inhibition of STAT1, it is verified that IFN1 can regulate the phagocytosis and immune factor expressions of thrombocytes through CRFB1/CRFB2/CRFB5 and JAK/STAT pathways. This lays the foundation for the study of IFN1 in the antibacterial pathway.

Phagocytosis is divided into opsonic phagocytosis (complement mediated or Ab mediated) and nonopsonic phagocytosis (PRR mediated) by the different initiating receptors (43). In the present study, IFN1 activates thrombocytes to produce various members of the complement system, including C1q, C1r, C3.3, C5aR, factor D, and CR1. This implies that IFN1 may play a role in regulating complement-mediated phagocytosis. The complement components exist in serum and tissue fluid and perform important biological functions such as cell lysis, cell adhesion, opsonization, immune regulation, and inflammatory responses (33). C3 is one of the key molecules in the complement system to exert biological effects. The content of C3 in serum is the most abundant, and it is cleaved into C3a and C3b in the physiological state. C3b is the main opsonin in the complement system, which can cover the microbes and then combine with the complement receptors on phagocytes, resulting in opsonic phagocytosis. Teleosts usually have multiple C3 genes; for example, zebrafish have eight C3 genes (51), grass carp have nine C3 genes (33), common carp have at least five C3 genes (52), and rainbow trout have at least three C3 genes, varying in amounts and thus functioning to varying degrees in the body (53). The contents of nine C3 genes in grass carp serum are different, with the highest being C3.1 and C3.3 being middling (54). In this study, the expression level of complement C3.3 showed the highest increase and was most abundant in the IFN1-stimulated thrombocytes. By Ab blockade of IFN receptors and inhibition of STAT1, we demonstrated that IFN1 promotes C3.3 and CR1 production via the CRFB1/CRFB2/CRFB5-JAK/STAT regulatory axis. Through blocking CR1 with Ab in thrombocytes, we further proved that the phagocytosis facilitated by IFN1 is mediated by complement components. C3.3 is induced and cleaved into C3.3b to capture bacteria; subsequently, C3.3b binds to complement receptor CR1 on the surface of the thrombocytes to participate in the opsonic phagocytosis. Here, we found a new mechanism by which IFN1 activates thrombocytes to express complement components involved in phagocytosis.

Complement components mainly distribute in serum. The activated complements can form a membrane attack complex to lyse and destroy bacteria (55). We wondered whether the complements induced by IFN1 in vivo can exert effective antibacterial function. Complements are heat sensitive; fish complements will lose activity after 45°C heat treatment for 30 min. We injected grass carp with the recombinant IFN1 protein and obtained sera. By heat treatment, we demonstrated that IFN1 endows serum with stronger bacteriolytic activity and that the bactericidal effect is exerted by the complements. IFN1 selectively promotes the rapid production of more C3.3 in serum. Unexpectedly, complement not only has bactericidal activity but also shows effective aggregation of bacteria. Classically, complements carry out the bacteriolysis, whereas the aggregation of bacteria in serum is caused by Abs in mammals. The present study indicates that complements possess both bacteriolysis and bacterial aggregation in teleosts. However, the mechanisms of bacterial aggregation by complements need to be further studied.

Does the antibacterial immune-regulatory function of IFN1 have physiological significance in bacterial infection? On the basis of the experimental result of blocking IFN1 in vivo, grass carp IFN1 can effectively reduce systemic bacterial infection at physiological levels. Meanwhile, injection of recombinant IFN1 protein exhibits an effective therapeutic role after bacterial infection. Such antibacterial effect not only kills bacteria and reduces tissue bacterial loads but also attenuates tissue edema and injury, finally increasing the survival rate. Therefore, high concentration of IFN1 plays a direct bactericidal role (15), whereas physiological concentration of IFN1 performs a systemic immune-regulatory function during bacterial infection. Cytokines with both direct bactericidal activity and immunomodulatory bactericidal activity have been studied in many mammals and teleosts. Mouse CCL28 has a broad-spectrum direct bactericidal activity in addition to its chemotactic effect (56). Grass carp CXCL20a not only can directly kill bacteria as an antibacterial protein but also has immunoregulatory functions such as chemotaxis and phagocytosis promotion (24). In previous studies, grass carp IFN1 and zebrafish IFN-φ1 demonstrated obviously protective effects against A. hydrophila and S. iniae infections in zebrafish (13, 15). Here, we shed light on the indirect immunoregulatory antibacterial role of IFN1 in bacterial infection. In contrast, mouse IFN-β stimulation does not significantly induce cytokines and C3 in mice and fails to enhance the antibacterial effect.

In conclusion, various cells containing PRRs recognize bacterial or viral microbe-associated molecular patterns and significantly induce IFN1. IFN1 exerts a physiological antibacterial effect in bacterial infection. High levels of IFN1 in blood activate the immune activity of blood cells, especially thrombocytes. IFN1 significantly promotes phagocytosis in a dose-dependent manner and expression of immune genes, including IL-1β, IL-2, MHC II, CD80/CD86, C3.3, and CR1. The thrombocyte phagocytosis enhanced by IFN1 is mediated by the CRFB1/CRFB2/CRFB5 receptor complex and the JAK/STAT, C3.3, and CR1 pathway. Complements demonstrate not only bacteriolysis but also aggregation of bacteria in teleost serum. The complement production enhanced by IFN1 is mediated by the IFN receptor complex and the JAK/STAT pathway. Meanwhile, the immune factors produced by thrombocytes can regulate other immune cells for antibacterial immune responses. These immunomodulatory effects of IFN1 in vivo can effectively reduce the tissue bacterial loads, edema, and injury, thus improving the survival rate of grass carp in bacterial infection (Fig. 11). This study, to our knowledge, revealed that type I IFN exerts an antibacterial function by complement-mediated phagocytosis, bacterial aggregation, bacteriolysis, and immune regulation, which enlightens the functional studies on IFN in bacterial infections.

The authors have no financial conflicts of interest.

We thank Dr. Xiaoling Liu for providing GFP-E. coli. We also appreciate Dr. Xun Xiao, Dr. Gailing Yuan, Rui Jiang, and Bo Liang for precious advice, helpful discussions, and friendly assistance in the experiments.