Socs3b regulates the development and function of innate immune cells in zebrafish

Abstract

Introduction:

Suppressor of cytokine signaling 3 (SOCS3) is a critical component of the negative feedback regulation that controls signaling by cytokines and other factors thereby ensuring that important processes such as hematopoiesis and inflammation occur at appropriate levels.

Methods:

To gain further insights into SOCS3 function, the zebrafish socs3b gene was investigated through analysis of a knockout line generated using CRISPR/Cas9-mediated genome editing.

Results:

Zebrafish socs3b knockout embryos displayed elevated numbers of neutrophils during primitive and definitive hematopoiesis but macrophage numbers were not altered. However, the absence of socs3b reduced neutrophil functionality but enhanced macrophage responses. Adult socs3b knockout zebrafish displayed reduced survival that correlated with an eye pathology involving extensive infiltration of neutrophils and macrophages along with immune cell dysregulation in other tissues.

Discussion:

These findings identify a conserved role for Socs3b in the regulation of neutrophil production and macrophage activation.

1 Introduction

The suppressor of cytokine signaling (SOCS) proteins regulate intracellular signaling induced by numerous extracellular regulators (1). Among the seven mammalian SOCS proteins, SOCS1-3 and the cytokine-inducible SH2-containing (CISH) protein are directly involved in the negative feedback control of cytokine receptor signaling, exerting a significant influence on processes such as hematopoiesis and innate immunity (1, 2). SOCS3 is highly expressed within the myeloid lineage, regulating both the differentiation and activation of neutrophils and macrophages (3, 4). Increased expression of SOCS3 has been observed in various models of chronic inflammation, corresponding with the degree of inflammation as well as the onset of various symptoms (5–7). However, decreased expression of SOCS3 is associated with the development of other inflammatory conditions, such as acute lung injury, hypersensitivity, and rheumatoid arthritis (8–10).

Global SOCS3 knockout mice exhibited embryonic lethality due to neutrophil-mediated inflammation, primarily within the placenta (11). This has been largely attributed to the role of SOCS3 in regulating signaling downstream of the granulocyte colony-stimulating factor receptor (G-CSFR) (12), with G-CSF stimulation of these mice resulting in uncontrolled inflammation and neutrophilic infiltration into various tissues, such as the liver, spleen and spine (4, 13). Hematopoietic specific deletion of SOCS3 also triggered a similar phenotype during late adulthood, with neutrophilia observed in both liver and lung, concurrent with elevated levels of IL-6 and G-CSF in these tissues (13).

The role of SOCS3 within the monocyte/macrophage lineage remains vague. In response to infections, activated macrophages displayed an upregulation of SOCS3 both in vitro and in vivo (14). Moreover, SOCS3-deficient macrophages showed increased expression of pro-inflammatory cytokines, such as IL-6, IL-1β and IL-12, as well as prolonged signaling through the JAK/STAT pathway in response to LPS (15). Furthermore, myeloid-specific deletion of SOCS3 exacerbated acute lung injury in response to LPS (7, 9). Lastly, the phagocytic abilities of macrophages were found to be upregulated following SOCS3 ablation, which has been attributed to enhanced IL-6 and IL-12 signaling (15, 16).

To better understand the in vivo function of SOCS3, this study generated a knockout mutant of the zebrafish socs3b gene (17). This mutant showed significantly elevated neutrophils throughout the life course although functional deficits were identified, while macrophages were more responsive to wounding. Adult socs3b mutant zebrafish developed a pathology of the eye, with infiltration of neutrophils into multiple tissues, suggesting a complex inflammatory disorder.

2 Methods

2.1 Zebrafish husbandry and line generation

Wildtype (WT), transgenic and mutant zebrafish were maintained in accordance with standard husbandry practices (18). To generate socs3b mutants, WT embryos were injected with ~1 pL 12.5 ng/μL guide RNA (gRNA)1 (5’-GUCCGAGCUGUCCCGCACC) and gRNA2 (5’-GGCGCACGCCGCCUUCAAAG) targeting socs3b and 100 ng/μL Cas9 mRNA (Sigma-Aldrich) at the 1 cell stage. Zebrafish were screened using PCR of genomic DNA extracted from fin clips using primers flanking the gRNA1 (5’-ACAGGGGGTGGGCAGAGC and 5’-GCTCACGGTGCTCCAGTAGAA) and gRNA2 (5’-GAGCGGGAAGGAGGCCAG and 5’-GCCGTTTTCACACTGAGCGT) target sites, followed by Sanger sequencing (Australian Genome Research Facility). Zebrafish carrying a suitable mutant allele were subsequently out-crossed with WT zebrafish for two generations. In order to minimize potential bias a mixed pool of embryos obtained from heterozygote mutant in-crosses were used, with genotyping performed post-analysis, as described (19). All experiments compared homozygous mutant zebrafish with WT siblings generated from the same cross and were performed in a blinded manner and repeated at least 3 times, with representative data shown. The mutant alleles were also crossed onto two transgenic lines Tg(mpx::GFP) (20) and Tg(mpeg1.1::GFP) (21).

2.2 Whole-mount in situ hybridization and Sudan black staining

Zebrafish embryos at relevant timepoints were fixed with 4% (w/v) paraformaldehyde (PFA) at 4°C prior to whole-mount in situ hybridization (WISH) using DIG-labelled anti-sense probes as previously described (22), with embryos younger than 22 hours post fertilization (hpf) dechorionated prior to fixing, or subjected to Sudan black staining (23). For probes that identified discrete cells (spi1, lyz, lcp1, mpeg1.1, mpx, csf3r, Sudan black), manual counting was performed across the embryos. For probes that showed staining in specific loci (hbbe1.1, rag1), the area was measured using ImageJ as the average from imaging both sides of the embryo (24). The region covering the majority of cells are shown in the respective figures.

2.3 Reverse-transcription polymerase chain reaction

Total RNA was extracted from zebrafish embryos and various adult tissues with either RNeasy Mini Kit (Qiagen) or Trisure (Bioline) according to the respective manufacturer’s protocol, depending on the nature of the sample. This was subjected to quantitative real-time reverse-transcriptase PCR (qRT²-PCR) with the primers listed in Table 1. Data were normalized relative to actb and fold-change calculated using the ΔCT method (31).

2.4 Ex vivo analyses

Adult zebrafish kidney cells were prepared in ice-cold phosphate buffered saline supplemented with 2 mM EDTA and 2% (v/v) fetal calf serum and passed through a 40 μm filter. For flow cytometry analysis, samples were analyzed on a FACSCanto II. Dead cells and doublets were removed by gating with lymphoid, erythroid, myeloid and precursor cells identified in a side scatter/forward scatter (SSC/FSC) plot and neutrophils and macrophages via GFP fluorescence using fish on the transgenic Tg(mpx::GFP) and Tg(mpeg1.1::GFP) backgrounds, respectively. Smears of kidney cells and adult blood were prepared using a cytospin and stained with Giemsa prior to differential counting and imaging using a Leica DME stereomicroscope and Olympus SC50 camera.

2.5 In vivo analyses

A wounding assay was utilized to identify relative neutrophil and macrophage migration using the transgenic lines and fluorescence microscopy (32). Embryos at 5 days post fertilization (dpf) were anaesthetized using 5 μg/mL benzocaine in system water. After the embryos stopped moving, an excision was made at the tip of the caudal fin with a number 21 scalpel. Embryos were then placed into 12 well plates containing 0.003% (w/v) PTU in system water and imaged at 2 hour intervals in the presence of 5 μg/mL benzocaine before being placed in fresh PTU solution to remove the benzocaine. Embryos were maintained on a heat block set at 28.5°C for the 24 hours of the assay and imaged on an Olympus MVX10 microscope with a DP74 camera using CellSens Dimension 1.6 software (Olympus) with UV excitation and a GFP filter for analysis. Cell numbers in the whole embryo prior to wounding and at the injury site across the time-course were manually determined. Cell areas were quantified with ImageJ using images obtained at the injury site during the peak of cell migration. The net rate of cell migration was calculated from the peak cell number divided by the corresponding time post-wounding, and the net rate of cell egress from the reduction from the peak until 24 hours post-wounding (hpw). In other experiments embryos were injected at the 1 cell stage with csf3a mRNA or at 3 dpf with lipopolysaccharide (LPS) as described (33).

2.6 Statistics

Statistical analyses were performed using GraphPad Prism version 8. Normality of data was tested using the D’Agostino-Pearson omnibus normality test and unequal variances using the F-test. For normally distributed data, the difference between two groups was analyzed for significance with a student t-test, with Welch’s correction if unequal variances were present. Where multiple t-tests were performed, the Bonferroni-Dunn method was used to correct for multiple comparisons. The difference between more than two groups was tested for significance using ordinary one-way ANOVA coupled with Tukey’s multiple comparison test. If data did not follow a normal distribution, the Mann-Whitney U test was performed. Survival and pathology incidence were visualized with a Kaplan-Meier graph, with the Gehan-Breslow-Wilcoxon test used to compare between different groups.

2.7 Sequence analysis

Protein sequences of human SOCS3 (NP_003946) as well as zebrafish Socs3a (NP_956244) and Socs3b (NP_998469) were obtained from GenBank and subjected to constraint-based multiple sequence analysis with COBALT (34).

3 Results

3.1 Generation of socs3b KO zebrafish

Both zebrafish Socs3a and Socs3b proteins share the same domain structure as human SOCS3 with very good overall conservation (Figure 1A), including many stretches of identical residues (Figure 1B). However, Socs3b shows higher conservation across the protein (Figures 1A, B) and so was chosen for further study. The socs3b gene also has the same exon structure as SOCS3 (17), with exon 2 targeted using two guide RNAs (gRNAs) directed at sequences encoding the N-terminus and SH2 domain (Figure 1C). These were injected along with Cas9 mRNA into embryos, which were raised to adulthood and out-crossed with wildtype (WT) fish. Screening of the resultant F1 fish identified a single mutant allele that impacted the socs3b reading frame. The carrier of this allele was again out-crossed before in-crossing to produce homozygous F3 mutant fish. Sequence analysis revealed that this allele, designated mdu24, possessed a complex insertion/deletion at both gRNA sites (Figure 1D). The first caused a frameshift resulting in 41 de novo residues after proline at position 15 followed by a stop codon, with the second occurring after this point and so not impacting the protein encoded, which represented a severely truncated knockout (KO) version of the Socs3b protein, lacking the SH2 and SOCS box domains (Figure 1E).

Figure 1

3.2 socs3b regulates the number of primitive and early definitive neutrophils

The potential effects of socs3b ablation on primitive hematopoiesis were assessed using WISH with lineage-specific markers at 22 hpf. No difference was observed between WT and KO embryos with spi1b, a marker of early myeloid progenitors (25) (Figures 2A–C), or the pan-leucocyte markers lyz (35) (Figures 2D–F) and lcp1 (36) (Figures 2G–I). Similarly, there was no difference between WT and KO embryos with the macrophage marker mpeg1.1 (37) (Figures 2J–L). However, the neutrophil population, identified using mpx (38), was significantly increased in KO embryos (Figures 2M–O). The number of cells expressing the alternate neutrophil marker csf3r (33) were elevated but not to a statistically significant level (Figures 2P–R).

Figure 2

Early definitive hematopoiesis was assessed using WISH at 5 dpf. A significant increase in mpx⁺ cells was again observed in KO compared to WT embryos (Figures 3A, B, E). There was also a significant elevation in cells expressing lcp1 (Figures 3C, D, F) or csf3r (Figures 3G–I), but not mpeg1.1 (Figures 3J–L). Analysis of mature erythrocytes using hbbe1.1 (39) (Figures 3M–O) and T lymphocytes using rag1 (40) (Figures 3P–R) revealed no differences between WT and KO embryos. To further investigate the potential impact of socs3b on innate immune cells fish carrying the KO allele were separately out-crossed with two transgenic lines Tg(mpx::GFP) (20) and Tg(mpeg1.1::GFP) (21) to facilitate visualization of neutrophils and macrophages, respectively. This confirmed a significant elevation in the number of mpx⁺ neutrophils (Figures 3S–U) but not mpeg1.1⁺ macrophages (Figures 3V–X) in KO compared to WT embryos.

Figure 3

3.3 socs3b regulates the functionality of neutrophils and macrophages

Further analysis of the Tg(mpx::GFP) embryos by light microscopy failed to detect any differences between WT and KO genotypes (Figures 4A, B), while fluorescence microscopy revealed similar distribution of neutrophils across the embryo (Figures 4C, D). To assess neutrophil maturation, 5 dpf embryos were stained with Sudan black as a marker of differentiation (23) (Figures 4E, F). The number of stained cells was again significantly higher in KO compared to WT embryos with no difference in staining intensity observed (Figure 4K), suggesting the increased neutrophils were mature. To further investigate neutrophil responses, lipopolysaccharide (LPS) was also injected into WT and KO embryos on the Tg(mpx::GFP) transgenic background to simulate bacterial infection (33). In WT embryos LPS induction was able to significantly increase the mpx⁺ neutrophil population, but this increase was not observed in KO embryos (Figures 4G–J, L).

Figure 4

Embryos derived from both transgenic lines were also subjected to a tail wounding assay at 5 dpf (Figure 5A). No difference was observed in the neutrophil response to injury between WT and KO embryos, with similar numbers and kinetics of mpx⁺ cells observed (Figures 5B, C). However, when this data was normalized with respect to total mpx⁺ cells, a significantly lower neutrophil response to wounding in KO embryos compared to WT was apparent (Figure 5D). In contrast, a significantly larger and more sustained response was seen for mpeg1.1⁺ macrophages in KO compared to WT embryos (Figures 5E, F). There was no difference between WT and KO embryos in relative size (Supplementary Figures 1A, B) or egress rate (Supplementary Figures 1C, D) for mpx⁺ or mpeg1.1⁺ cells during wounding, but there was a significant increase in the rate of migration for mpeg1.1⁺ (Supplementary Figure 1F) but not mpx⁺ (Supplementary Figure 1E) cells in KO embryos. Gene expression analysis revealed a significant increase in the inflammatory markers cxc8la, il1b, il6 and tgfb1b (41, 42) in both WT and KO embryos after wounding but two markers of inflammatory macrophages ccr2 and cxcr3.2 (42) were significantly upregulated only in KO embryos (Figure 5G). Comparing gene expression between genotypes at the various timepoints revealed elevation of mpeg1.1, ccr2 and il6 basally and all genes by 8 hpw in KO compared to WT fish (Supplementary Figure 2).

Figure 5

3.4 socs3b impacts steady-state adult myeloid populations in zebrafish

Adult KO zebrafish had a major sex bias, with females drastically underrepresented, meaning it was only feasible to characterize adult male KO zebrafish. These were significantly smaller than male WT zebrafish at 5 months post fertilization (mpf), but otherwise anatomically normal (Figures 6A–C). Gene expression analysis of the kidney, which is the equivalent of mammalian bone marrow with respect to adult hematopoiesis (43), revealed a significant and specific increase in the neutrophil marker mpx in socs3b KO compared to WT fish, but not markers of macrophages (mpeg1.1) (21), T cells (tcra, cd4, cd8) (44), B cells (cd79a) (45), NK cells (nkl1.3) (46), thrombocytes (tpor) (47), RBC (hbaa1.1) (47), or precursors (cmyb, scl, ikzf1, runx1, gata1a) (48–50) (Figure 6D). Forward and side scatter analysis of kidney cells also identified a significantly larger myeloid cell compartment in KO zebrafish (Figures 6E–G). Moreover, analysis of fish on the Tg(mpx::GFP) and Tg(mpeg1.1::GFP) backgrounds revealed a significant increase in kidney neutrophils (Figures 6H–J) – but not macrophages (Figures 6K–M) – in KO compared to WT fish. Analysis of kidney smears confirmed a specific increase in mature neutrophils in KO fish that were morphologically indistinguishable from WT (Figures 6N–P), but this increase was not reflected in blood smears in which neutrophil numbers were not significantly different between genotypes (Figures 6Q–S).

Figure 6

3.5 socs3b KO zebrafish suffer from chronic inflammation during adulthood

A significant proportion of KO zebrafish developed an eye pathology, with these fish eventually becoming emaciated (Figure 7A). Affected individuals displayed significant tissue growth on the surface of the eye, in most cases covering the pupil and resulting in the development of so-called ‘bug eye’ (51) (Figures 7A–C). This tissue contained both neutrophils and macrophages as revealed in Tg(mpx::GFP) (Figure 7B) and Tg(mpeg1.1::GFP) (Figure 7C) lines, respectively. This eye pathology was first evident at around 1 mpf and reached almost 30% by 4 mpf (Figure 7D), and strongly correlated with early mortality in these fish (Figure 7E). Gene expression analysis of whole eyes confirmed an upregulation of neutrophil (mpx) (10) and macrophage (mpeg1.1) (37) markers in symptomatic KO fish compared to WT fish, as well as one T cell marker (cd4) (52), although the other T cell marker (cd8) and those for B cells (ighm) (53) and NK cells (nkl.3) (46) were not significantly altered (Figure 7F). Several inflammatory macrophage markers (cxcr3.2, tnfa, il1b) (42) were upregulated, along with inflammatory cytokines il4, il6, il21 (54) and ifng (55), but not cxcr4b, ccr2, tgfb1b or csf3b (Figure 7F). The gene expression profiles of various tissues extracted from normal WT and both symptomatic and asymptomatic KO adults were also assessed (Supplementary Figure 3). The neutrophil marker mpx was found to be significantly upregulated in the spleen and liver of symptomatic KO compared to WT, and compared to asymptomatic KO for liver, but was down-regulated in muscle (Supplementary Figures 3A-C). No significant change was observed in macrophage marker mpeg1.1 in any of these tissues (Supplementary Figures 3A–D). The lymphocyte marker cd8 was also significantly upregulated in both the liver and spleen of symptomatic and asymptomatic KO compared to WT, with cd4 upregulated in asymptomatic KO only for these tissues (Supplementary Figures 3A, B).

Figure 7

4 Discussion

SOCS3 is strongly implicated in the regulation of innate immune cell production and function (56, 57), with its disruption associated with a range of inflammatory conditions (1). Zebrafish possess two paralogous genes, socs3a and socs3b, the encoded proteins of which show conservation of functional domains (17) and activation downstream of Stat3 (58). To provide further insight into the role of SOCS3 in innate immunity, CRISPR/Cas9-mediated genome editing was used to generate a socs3b KO mutant that lacked all functional domains. Fish harboring this mutation were viable into adulthood, in contrast to global Socs3 knockout mice that suffered from embryonic lethality (11, 59), enabling analysis across the full life-course. These socs3b KO fish displayed enhanced neutrophil production, altered macrophage function, and developed an inflammatory phenotype during adulthood associated with a striking eye pathology and extensive neutrophil infiltration.

Zebrafish socs3b mutants showed a significant enhancement in the number of differentiated neutrophils during both the primitive and early definitive waves of hematopoiesis in embryos and extending to steady-state levels across multiple adult tissues. Morpholino-mediated knockdown of socs3b also elicited an increase in embryonic neutrophil numbers (data not shown) corroborating the results from the mutant allele. This is consistent with the neutrophilia observed in Socs3 knockout mice, which included infiltration into multiple organs (4). Moreover, elevated socs3b mRNA levels observed in zebrafish Tet mutants correlated with defective neutrophil maturation (60) identifying Socs3b as a crucial regulator of neutrophil production. Signaling by G-CSFR plays a key role in this process (33), with Socs3 knockout mice being hypersensitive to G-CSF stimulation (4, 13), suggesting unrestrained G-CSFR signaling as the underlying cause of the neutrophilia in these animals. Zebrafish G-CSFR has a conserved role in neutrophil production and function (33, 61, 62) and possesses SOCS3 binding sites in its intracellular domain like its mammalian homologues (33), with enforced G-CSF expression leading to strong socs3b induction (Supplementary Figure 4A). Together this indicates that dysregulated G-CSFR signaling likely also contributes to the increased neutrophils in zebrafish socs3b mutants.

Macrophage numbers were not altered in socs3b KO mutants, again consistent with mouse Socs3 knockouts (4, 13). However, embryonic macrophages were more responsive to injury in socs3b KO mutants, with increased number and sustained retention observed at the site of injury. This occurred concurrently with upregulation of chemokine receptors cxcr3.2 and ccr2, both of which are synonymous with macrophage recruitment to injury where they play crucial roles in tissue repair (63–65). A significant increase in CD4+ and CD8+ T cells was also observed in the spleen and liver of socs3b mutants. This also has some precedence in mice, where SOCS3 has been demonstrated to regulate CD8+ T cell proliferation mediated by IL-6 and IL-27 (66). In contrast, neutrophil functionality was reduced in socs3b KO fish.

The zebrafish socs3b mutants developed an eye pathology that was associated with increased infiltration of neutrophils, macrophages and T-lymphocytes, as well as elevated expression of the genes encoding the cytokines IL-1β, TNF-α and IFN-γ. The eye pathology was reminiscent of autoimmune uveitis, showing similar characteristics including upregulation of TNF-α (67). Interestingly, T cell specific Socs3 deletion protected mice from both chronic and acute experimental autoimmune uveitis (EAU) through an increase in regulatory T cells that produced higher levels of IL-10 (68). In contrast, myeloid specific Socs3 deletion exacerbated the development of inflammation-mediated retinal degradation in EUA, with increased neutrophil infiltration and enhanced levels of IL-1β, TNF-α and IFN-γ (69). These mice also exhibited worsened experimental autoimmune encephalomyelitis (EAE), with infiltration of neutrophils, B-cells and Th1 cells into the brain, along with demyelination and increased levels of IFN-γ, IL-6 and IL-17, ultimately resulting in earlier lethality (15). Neutrophils in these mice showed heightened STAT3 activation in response to G-CSF, resulting in increased production of reactive oxygen species (ROS), with ablation of G-CSF ameliorating the severity of EAE suggesting this cytokine was responsible for the severe inflammation (70). Collectively, this suggests a role for excessive G-CSFR signaling in the neutrophil lineage in the eye pathology. Increased expression of socs3b has been associated with optic nerve pathology (71). It was also upregulated following optic nerve injury, but knockdown of socs3b had no effect on axon regeneration (30), suggesting a role outside the optic nerve. However, SOCS3 is also expressed in photoreceptor cells and plays a protective role in these cells during inflammation by suppressing STAT3 activation (72), indicating a more complex etiology is likely.

Collectively our observations are consistent with chronic systemic inflammation in the KO fish. Whether this is due to the increased number of neutrophils, their altered functionality, the enhanced responsiveness of macrophages or a combination of these remains to be determined, but could be explored by ablation of specific cell populations such as using NTR/metronidazole. The neutrophil dysregulation is particularly intriguing, since their maturation appears normal but their responsiveness is blunted. One key aspect may relate to mobilization/migration with decreased neutrophil migration to injury in embryos, but also normal circulating neutrophil numbers despite increased stores in the kidney. However, other critical neutrophil functions – such as phagocytosis, NETosis and ROS production – may also be impacted, any of which could contribute to the phenotypes observed. Further close examination of the eye pathology and the response to injury in adults would likely provide useful insights.

Multiple SOCS proteins have been implicated in the development of autoimmune and inflammatory diseases. Dysregulated expression of SOCS3 has been frequently observed in various forms of inflammatory disorders, such as rheumatoid arthritis and Crohn’s disease (73, 74), with SOCS1 haploinsufficiency shown to predispose to early onset autoimmune disease (75). CISH knockout mice showed increased susceptibility to experimental allergic asthma (76) and EAE (77), while SOCS5 was preferentially expressed in the retina and significantly upregulated during the development and resolution EAU (78). As a result therapeutic strategies targeting SOCS proteins have been developed to treat relevant diseases. For example, mimetic SOCS1 peptides were able to reduce intraocular inflammation and EAU development in mice by suppressing the activities of IFN-γ and TNF-α and thereby preventing ocular damage (79, 80). Cell-penetrating SOCS3 forms have also been shown to be efficacious in inhibiting inflammation, with inhibition of IFN-γ and TNF-α activity again observed (81, 82). Our zebrafish model is ideally set up to further explore potential therapeutic agents for SOCS3, which could be readily extended to other SOCS proteins. Further studies could also explore the role of Socs3a, with evidence that it may compensate following the loss of Socs3b and/or act in parallel (Supplementary Figures 4B, C).

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