Tnfrsf1b Protects Zebrafish Against Klebsiella pneumoniae Infection

Introduction

Tumor necrosis factor-alpha (TNF-α), a member of the TNF superfamily, can be produced by many immune cells under pathologically related conditions and plays different roles.^1–4 For example, TNF-α is involved in many biological processes, including autoimmunity,⁴ late tumor development,⁵ and arthritis.⁶ TNFR1 and tnfrsf1b are TNF-α signal receptors. TNFR1 is expressed in most cell types; however, tnfrsf1b is restricted primarily to endothelial, epithelial, and subsets of immune cells.⁷ The TNFR1 signaling pathway is involved in inflammation and apoptosis pathways, whereas the tnfrsf1b signaling pathway is involved in the anti-inflammatory response and initiation of apoptosis.⁸ Inhibiting TNFR1 alters autoimmune-related homeostasis; however, tnfrsf1b initiates repair after injury⁹ and participates in developmental processes, such as heart development.¹⁰ Recently, tnfrsf1b was reported to play a vital role in treating many diseases as a new drug candidate,¹¹ but the functional mechanism of tnfrsf1b remains unclear.

Klebsiella (K). pneumoniae, an opportunistic pathogen, is widely distributed in nature and in almost all hospitals.¹² Under normal circumstances, K. pneumoniae does not result in pathological infections;¹³ however, when the body’s immunity is low, K. pneumoniae infection can cause pneumonia, urinary tract infection, liver abscess, bacteremia, and other symptoms.¹³ K. pneumoniae infection can become serious and even life-threatening, especially for patients in the intensive care unit or older people.¹⁴ Unfortunately, there are still no effective drugs or methods to treat this infection. Previous research has focused on the virulence of pathogenic bacteria and the response of the host immune system.¹⁵ Pro-inflammatory responses have been found to play important roles in the elimination of K. pneumoniae.¹⁶ Hu and Cai reported that immunodeficient mice can be easily infected by K. pneumoniae.¹⁷ The clearance of K. pneumoniae is significantly reduced and the susceptibility to infection is increased in mice deficient in TNFR1 and CXCL15.^18,19

In recent years, the clinical significance of K. pneumoniae has dramatically increased due to the emergence of hypervirulent strains, such as NTUH-K2044, which are associated with liver abscess, endophthalmitis, and disseminated infections even in otherwise healthy individuals. These strains often exhibit enhanced capsule production, increased invasiveness, and resistance to phagocytosis, leading to poor clinical outcomes. At the same time, the global rise of antimicrobial resistance in K. pneumoniae, including carbapenem-resistant and extended-spectrum β-lactamase (ESBL)-producing isolates, has created serious challenges in treatment. The dual threat of hypervirulence and multidrug resistance makes K. pneumoniae a pathogen of urgent clinical concern, underscoring the need to better understand host–pathogen interactions and identify new candidate targets such as tnfrsf1b.

The zebrafish is a model organism²⁰ that has been widely used to study the pathogenicity, virulence, and drug resistance of bacteria, as well as host reactions to bacterial infection.^21–24 Our previous study demonstrated that K. pneumoniae infection leads to intestinal inflammation and microbial dysbiosis in adult zebrafish, using clinically isolated strains with varying virulence levels.²⁵ However, the host genetic determinants contributing to susceptibility or resistance to K. pneumoniae infection remained largely unexplored. The current study is fundamentally distinct from the previous work in the following aspects: (1) Mechanistic Focus: While the earlier study centered on comparative pathophysiological outcomes caused by different K. pneumoniae strains, the current research explores the functional role of tnfrsf1b signaling in host-pathogen interactions. (2) Genetic Manipulation: We employed a CRISPR/Cas9-generated tnfrsf1b-knockout zebrafish line, which allows us to dissect the immunological role of tnfrsf1b in infection susceptibility and inflammatory regulation. (3) Therapeutic Relevance: This study demonstrates the protective effects of tnfrsf1b-capped mRNA and identifies tnfrsf1b as a potential therapeutic target against hypervirulent K. pneumoniae. (4) Functional Rescue and Pharmacological Inhibition: We further validate our findings by both genetic rescue (mRNA injection) and pharmacological inhibition (R7050), revealing mechanistic depth that was not addressed in the previous publication. The TNF-α receptors TNFR1 and tnfrsf1b both exist in zebrafish, but their function is still not completely understood. In this study, the tnfrsf1b gene in zebrafish was knocked-out using CRISPR/Cas9 technology, and then the wild-type (WT) and tnfrsf1b^−/− zebrafish were infected with K. pneumoniae strain NTUH-K2044 to study the function of tnfrsf1b in the interaction of K. pneumoniae and host. The immune factors and immune cells and partial gene expression patterns were investigated. This work may provide a theoretical basis for the clinical treatment of K. pneumoniae infection.

Materials and Methods

Fish Maintenance

All zebrafish embryo experiments were performed in compliance with national and institutional animal welfare regulations. Adult AB-strain zebrafish were maintained in a recirculating water system under a controlled 14-hour light and 10-hour dark cycle at 28 °C, and fed three times daily. For embryo collection, male and female fish were placed together in mating tanks the night before, and fertilization occurred within an hour of light onset the following morning. Collected embryos were incubated in 10 cm Petri dishes containing egg water supplemented with 0.3% methylene blue and maintained in a 28 °C incubator under the same 14/10 h light/dark schedule. Embryos were sampled at 4–6 days post-fertilization (dpf) for whole-mount Alcian blue staining. At 5 dpf, additional samples were harvested for behavioral testing and qRT-PCR analysis.

Generation of tnfrsf1b Mutant Zebrafish

The tnfrsf1b knockout zebrafish line was established using CRISPR-Cas9 genome editing. A target sequence within the first exon was selected, and the corresponding guide RNA (gRNA) was synthesized based on established protocols. To induce mutations, zebrafish embryos at the one-cell stage were co-injected with 100 pg of gRNA and 300 pg of Cas9-capped mRNA. Following injection, embryos were cultured at 28.5 °C in E3 medium (composed of 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, and 0.33 mM MgSO₄). At 2 days post-fertilization (dpf), genomic DNA was extracted from three pools of 10 embryos each. A 194 bp DNA fragment encompassing the target region was amplified by PCR (primer sequences provided in Table S1). To assess mutation efficiency, 7 μL of PCR product was digested with T7 Endonuclease I (New England Biolabs) at 37 °C for 3 hours, and cleavage efficiency was quantified using NIH ImageJ software. PCR amplicons were cloned into the pMD-19T vector (Takara) for Sanger sequencing. To confirm germline transmission, founder (F0) zebrafish were raised to adulthood and crossed with wild-type fish. Genomic DNA from 10 F1 embryos per cross was analyzed by enzymatic digestion (data not shown). Sibling F1 carriers of heritable mutations were grown to maturity, and individual mutants were re-verified via PCR and sequencing of fin-derived DNA. Homozygous tnfrsf1b mutants were obtained by mating male and female F1 fish carrying the same deletion. A stable mutant line harboring a 5 bp deletion in tnfrsf1b was successfully established.

Bacterial Immersion Experiments

Bacterial cultures were grown to the logarithmic phase, and the concentration of the suspension was standardized by measuring OD600. A conversion curve between OD600 and colony-forming units (CFU) was established in preliminary experiments, and the immersion infection dose was adjusted to 5 × 10⁶ CFU/mL accordingly. To further assess infection progression, bacterial loads in zebrafish larvae were determined at designated time points post-infection by homogenizing groups of 10 larvae, plating serial dilutions on LB agar, and counting colonies after overnight incubation at 37 °C.

The K. pneumoniae immersion infection model was modified from previously established protocols. Bacteria were cultured to the logarithmic growth phase (OD600 = 0.4–0.6), then collected by centrifugation, washed, and resuspended in sterile E3 medium. At 3 days post-fertilization (dpf), both wild-type and tnfrsf1b mutant zebrafish larvae were rinsed with fresh E3 medium and allocated into sterile 6-well plates, with 10 larvae placed in each well. Each well was filled with 8 mL of bacterial suspension at a concentration of 5 × 10⁶ CFU/mL. Larvae were maintained in this suspension at 28 °C for 72 hours. After incubation, the larvae were collected and processed for subsequent experimental procedures. Each immersion assay was independently repeated six times, with a total of 75 larvae assigned to each experimental group.

Staining of Neutrophils and Macrophages in Zebrafish

Zebrafish larvae were initially fixed in 2% paraformaldehyde and gently agitated for 2 hours, followed by overnight staining with Sudan Black B (Solarbio, USA), a lipid-based dye that specifically labels neutrophil granules. After an 8-hour co-incubation with bacteria, 10 larvae from each group were fixed in 4% paraformaldehyde, rinsed with phosphate-buffered saline containing Tween, and again stained with Sudan Black B to visualize neutrophils. Following a 70% ethanol wash, neutrophil recruitment was examined microscopically. The preparation of the staining solution followed a previously published protocol.

For assessing macrophage phagocytic activity, groups of 10 zebrafish larvae were placed in 12-well plates containing 2.5 mg/L neutral red dye solution (Solarbio, China). This dye accumulates in macrophages as red puncta due to active endocytosis. After 8 hours of bacterial exposure, the larvae were anesthetized, and macrophage phagocytosis of neutral red was observed under a microscope.

RNA Isolation, cDNA Synthesis, and qRT-PCR

Total RNA was isolated from pools of over 25 zebrafish larvae using TRIzol reagent (Invitrogen), followed by reverse transcription into complementary DNA (cDNA) using Superscript III Reverse Transcriptase (Invitrogen, USA). Quantitative real-time PCR (qRT-PCR) was conducted on an ABI Step-One Plus system employing the SYBR Green detection method (TaKaRa, Dalian, China). The thermal cycling conditions included 40 amplification cycles consisting of denaturation at 95 °C for 10 seconds and annealing/extension at 58 °C for 30 seconds. Each reaction was run in triplicate using independently collected biological replicates. Gene expression levels were normalized to the reference gene β-actin, and relative quantification was determined using the 2^–ΔΔCT method. Primers used for qRT-PCR (listed in Table S1) were designed to span exon-exon junctions using Primer Express 5.0 software and were evaluated for secondary structures, such as self-dimers and hairpins, using Oligo Analyzer.

Inflammatory Factor Analysis by Enzyme-Linked Immunosorbent Assay (ELISA)

A total of 50 zebrafish larvae were homogenized in 0.5 mL of 0.9% sodium chloride solution using a tissue homogenizer. The resulting homogenate was centrifuged at 3000 rpm for 10 minutes at 4 °C, and the supernatant was collected for subsequent cytokine analysis, including interleukin-1α (IL-1α), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α). Enzyme-linked immunosorbent assay (ELISA) was conducted following the manufacturer’s protocols (Jianchengbio, Nanjing, China). Each sample and standard was analyzed in triplicate to ensure accuracy.

Statistical Analyses

All data are expressed as mean ± standard deviation (SD). Statistical comparisons between two groups were performed using Student’s t-test, while multiple group comparisons were analyzed by one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons. Each experiment was independently repeated a minimum of three times. A p value less than 0.05 was regarded as statistically significant.

Results

Bioinformatics of Zebrafish tnfrsf1b and Construction of Mutation

We first analyzed the protein sequences of zebrafish tnfrsf1b, comparing with the homologous protein from human and mice. Although zebrafish tnfrsf1b shared only 24.15% and 21.8% amino acid sequence identity with the homologues of human and mice, respectively (Figure 1A), the functional domains of the three proteins are conserved, composed of four TNFR domains and a transmembrane region (Figure 1B).

Figure 1 Evolutionary conservation and mutant of zebrafish tnfrs1b. (A) Conservative comparison of human, mouse, and zebrafish amino acid sequences. (B) Conservation type comparison of the functional domain of human, mouse, and zebrafish tnfrs1b. (C) The structure of zebrafish tnfrs1b gene and the acquisition of mutants. Red box indicates TNFR Domain 1, yellow box indicates TNFR Domain 2, green box indicates TNFR Domain 3, the purple box indicates TNFR Domain 4, and blue box represents the Transmembrane Zone.

The zebrafish tnfrsf1b gene contains 9 exons (Figure 1C). To construct a mutant strain with complete gene inactivation, we designed the gRNA site in the first exon region of tnfrsf1b gene and obtained a homozygous mutant with 5 bases depletion (Figure 1C).

Phenotype Analysis of Zebrafish tnfrsf1b Mutant

We first investigated the impacts of tnfrsf1b knockout on development by observing egg production, fertilization and hatchling. The homozygous zebrafish tnfrs1b mutant reduced egg production of female zebrafish (Figure 2A). Fertilization rate of the eggs also showed a ~20% decrease (Figure 2B). Hatchling rate, a key step for development, was also affected by absence of tnfrsf1b, while introduction of tnfrsf1b mRNA also partially reversed the impacts by mutation (Figure 2C–H).

Figure 2 Phenotype analysis of zebrafish tnfrs1b mutant. (A–C) Egg production, fertilization rate, and hatching rate of zebrafish tnfrs1b mutant. (D) Mortality rate of zebrafish tnfrs1b mutant at 72 and 96hpf; (E and F) Deformed appearance and ratio of zebrafish tnfrs1b mutant. (G–I) Mortality, hatching rate, and deformity rate of rescue zebrafish tnfrs1b mutant. * P < 0.05, ** P < 0.01, *** P < 0.001.

In addition, we observed the impacts of tnfrsf1b knockout on death and malformation rate of larvae fish. tnfrsf1b mutation caused significant increase of death rate at 96 hpf, which could be partly reversed by introduction of tnfrsf1b mRNA (Figure 2D and G). The tnfrs1b mutant exhibited a malformation rate is as high as 34% at 72hpf. The impacts of mutation could also be reversed by introduction of tnfrsf1b mRNA from 48–96 hpf (Figure 2E–I). The results indicate that the tnfrs1b mutant has obvious reproductive and developmental defected phenotype.

Zebrafish tnfrsf1b Mutant Has an Evident Immune Dysfunction Phenotype

Neutral red staining showed that the number of macrophages in the tnfrsf1b mutant significantly increased compared with that in the WT (Figure 3A and B). Sudan black staining showed that the number of neutrophils in the tnfrsf1b mutant also significantly increased (Figure 3C and D). Using qRT-PCR, we found that the mRNA level of tnf-α, il-1β, and il-6 was significantly upregulated, while the mRNA level of tnf-β, ccl2, ccl3, cxcl2, and ifng1 did not change significantly (Figure 3E). ELISA results showed that the protein expression of TNF-α and IL-1β was significantly increased in the tnfrsf1b mutant (Figure 3F). These results indicate that there is an evident immune dysfunction phenotype in the tnfrsf1b mutant.

Figure 3 Deletion of zebrafish tnfrsf1b leads to dysfunction. (A) Zebrafish tnfrsf1b mutant macrophage staining. (B) Statistics of optical intensity of macrophages. (C) Zebrafish tnfrsf1b mutant neutrophil staining. (D) Optical density statistics of neutrophils. (E) Expression of immune-related genes in the zebrafish tnfrsf1b mutant. (F) ELISA to detect the protein expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in tnfrsf1b mutants. Data are expressed as mean ± standard deviation (SD),* P < 0.05, ** P < 0.01, *** P < 0.001 compared with the WT group. Abbreviation: WT, wild type.

Treatment of WT Zebrafish with the TNFR Inhibitor R7050 Leads to Evident Immune Dysfunction

R7050 is an inhibitor of TNFR, which can inhibit the activities of TNFR1 and tnfrsf1b. After treating WT zebrafish with R7050, the malformation rate of zebrafish was significantly increased in the 72 hpf (Figure 4A). ELISA showed that tnfrsf1b protein expression was inhibited, as expected. Furthermore, the number of macrophages (Figure 4B and C) and neutrophils (Figure 4D and E) was also significantly increased. qRT-PCR revealed that tnfa, tnb, il1b, il6, ccl2, ccl3, and cxcl2 were significantly upregulated (Figure 4F). In addition, the protein expression of TNF-α and IL-1β increased significantly after R7050 treatment (Figure 4G). These results indicate that the treatment of WT zebrafish with the TNFR inhibitor R7050 leads to significant immune dysfunction.

Figure 4 Treatment of wild-type (WT) zebrafish with TNFR inhibitor R7050 results in evident immunodeficiency. (A) Treatment of WT zebrafish with TNFR inhibitor R7050 resulted in evident malformations at 72 and 96 hours post-fertilization (hpf). (B) Changes in macrophages in WT zebrafish treated with R7050. (C) Statistical analysis of macrophages. (D) Changes in neutrophils in WT zebrafish treated with R7050. (E) Statistical analysis of neutrophils. (F) Changes in immune-related genes in WT zebrafish treated with R7050. (G) ELISA was used to detect the changes in tumor necrosis factor (TNF)-α and interleukin (IL)-1β protein concentrations in WT zebrafish treated with R7050. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the WT group.

Infection of Zebrafish with K. pneumoniae Strain NTUH-K2044 Enhances Immune Dysfunction in tnfrsf1b Mutant

WT and tnfrsf1b mutants were infected with K. pneumoniae NTUH-K2044. There was no significant difference in the proportion of deformities in the tnfrsf1b mutants in the presence and absence of K. pneumoniae (Figure 5A). After treating the WT with the same concentration of K. pneumoniae, there was no significant increase in mortality at 72 and 96 hpf compared with the control group; however, compared with that of tnfrsf1b mutants not treated with K. pneumoniae, the mortality rate of tnfrsf1b mutants treated with K. pneumoniae increased significantly at the same time point (Figure 5B).

Figure 5 Treatment of tnfrsf1b mutants with K. pneumoniae strain K2044 enhances immune damage caused by tnfrsf1b deficiency. Changes in the deformity rate (A) and mortality (B) of K. pneumoniae strain K2044-treated tnfrsf1b mutants. (C) Staining of macrophages after treatment with K. pneumoniae strain K2044 in tnfrsf1b mutants. (D) Staining of neutrophils after treatment with K. pneumoniae strain K2044 in tnfrsf1b mutants. (E) Statistics of macrophages. (F) Statistics of neutrophils. (G) K. pneumoniae strain K2044 treatment of tnfrsf1b mutants resulted in immune-related gene expression changes. (H) ELISA was used to detect the changes of tumor necrosis factor (TNF)-α and interleukin (IL)-1β protein concentrations after treatment of tnfrsf1b mutants with K. pneumoniae strain K2044. * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviations: WT, wild type; hpf, hours post-fertilization; ns, not significant.

As shown in Figure 5C–F, K. pneumoniae treatment increased the number of neutrophils and macrophages, with a more severe phenotype observed in the tnfrsf1b mutant both in WT and mutant zebrafish. However, the increase in the tnfrsf1b mutant was more considerable. qRT-PCR results showed that K. pneumoniae treatment of the tnfrsf1b mutants significantly increased the mRNA level of inflammation-related genes, such as il-1β, il-8, ccl2, ccl3, and cxcl2 (Figure 5G). Furthermore, ELISA showed that the protein concentrations of TNF-α and IL-1β significantly increased in K. pneumoniae-treated WT and tnfrsf1b mutant zebrafish (Figure 5H).

R7050-Treated WT Zebrafish Subjected to K. pneumoniae NTUH-K2044 Treatment Show a Phenotype Similar to That of tnfrsf1b Mutants Treated with K. pneumoniae

WT zebrafish pre-treated with R7050 were infected with K. pneumoniae. The malformation rate (Figure 6A) and the number of macrophages and neutrophils (Figure 6B–E) increased significantly compared with those in WT zebrafish not treated with R7050. Furthermore, the mRNA level of il-1β, il-8, ccl2, ccl3, cxcl2, and ifng1 was significantly increased after the addition of R7050. K. pneumoniae added to R7050-pre-treated WT zebrafish resulted in a significant downregulation in the expression of cxcl2 and ifng1, but no significant differences in other detected genes (Figure 6F), compared to that in zebrafish treated with R7050 alone. Furthermore, ELISA analysis revealed that the protein concentrations of TNF-α and IL-1β were significantly increased in WT zebrafish treated with R7050 following K. pneumoniae infection, compared to controls (Figure 6G). These results further confirm that combined pharmacological inhibition of TNFR signaling and bacterial challenge exacerbates immune dysfunction.

Figure 6 Addition of Klebsiella pneumoniae strain K2044 after treatment of wild-type (WT) zebrafish with R7050 resulted in a more severe phenotype. (A) Changes in the malformation rate of K. pneumoniae strain K2044 after treatment of WT zebrafish with R7050. (B and C) Changes in macrophages after the addition of K. pneumoniae strain K2044 following treatment with R7050 in WT zebrafish. (D and E) Changes in neutrophil granulocytes in WT zebrafish treated with R7050 after adding K. pneumoniae strain K2044. (F) Changes in immune-related genes after adding K. pneumoniae strain K2044 to WT zebrafish treated with R7050. (G) ELISA was used to detect the changes of tumor necrosis factor (TNF)-α and interleukin (IL)-1β protein concentrations after adding K. pneumoniae strain K2044 to WT zebrafish treated with R7050. Hpf, hours post fertilization. * P < 0.05, ** P < 0.01, *** P < 0.001.

tnfrsf1b-Capped mRNA Can Rescue the Phenotype of tnfrsf1b Mutants and Protect Zebrafish Against Damage Caused by K. pneumoniae

tnfrsf1b-capped mRNA was injected into single-celled WT and tnfrsf1b mutants. Compared with that in WT zebrafish, tnfrsf1b-capped mRNA significantly inhibited the increase in macrophages and neutrophils in tnfrsf1b mutants (Figure 7A–D).

Figure 7 Tnfrsf1b-capped mRNA can rescue immunodeficiency caused by tnfrsf1b deletion. Changes in macrophages (A and B), neutrophils (C and D), mortality (E), and malformation rate (F) after injection of tnfrsf1b-capped mRNA into tnfrsf1b mutants. Changes in macrophages (G and H) and neutrophils (I and J) after injection of tnfrsf1b -mRNA into wild-type (WT) and tnfrsf1b mutant zebrafish. * P < 0.05, ** P < 0.01, *** P < 0.001. Abbreviation: Hpf, hours post-fertilization.

To verify whether tnfrsf1b-capped mRNA had a protective effect during K. pneumoniae infection, tnfrsf1b-capped mRNA was then injected into WT and tnfrsf1b mutants; when the embryos reached 3 dpf, NTUH-K2044 was added. The results showed that tnfrsf1b -capped mRNA significantly rescued the malformation rate and death rate caused by NTUH-K2044 in the tnfrsf1b mutants compared with the WT (Figure 7E and F). In addition, tnfrsf1b-capped mRNA significantly reduced the number of neutrophils and macrophages following NTUH-K2044 infection in the tnfrsf1b mutants (Figure 7G–J).

Discussion

In this study, we first successfully obtained a zebrafish tnfrsf1b mutant with a 5-bp deletion using CRISPR/Cas9 technology. The tnfrsf1b mutant showed a clear immunodeficiency phenotype, manifested as an increase in the number of macrophages and neutrophils and an increase in levels of some inflammatory factors. Infecting the tnfrsf1b mutant with K. pneumoniae increased the malformation rate, mortality rate, and immune responses. tnfrsf1b-capped mRNA could rescue this phenotype and protected zebrafish against K. pneumoniae infection. The results indicated that tnfrsf1b plays a role in the development of zebrafish. Smith et al found that TNF-α/tnfrsf1b signaling is required for glial ensheathment at the dorsal root entry zone and that tnfrsf1b is also involved in the regulation of bone by the proteasome. In this study, there was a significant increase in the levels of immune cells and cytokines in the tnfrsf1b mutant. Macrophages are mainly fixed cells or free cells that phagocytose cell fragments and pathogens (that is, phagocytosis and digestion) and activate lymphocytes or other immune cells to respond to pathogens.²⁶ Neutrophils exert chemotactic, phagocytic, and bactericidal effects.²⁷ Under normal conditions, the increase in these two types of cells indicates that tnfrsf1b reduction may be caused by the susceptibility to some microorganisms in the environment. Therefore, the establishment of the tnfrsf1b mutant can be used as an immune disorder zebrafish model to study its related immune homeostasis.

The tnfrsf1b mutant causes an increased susceptibility to K. pneumoniae. Studies in mice have found that the deletion of many immune genes can increase the susceptibility to K. pneumoniae infection.^18,28 Since tnfrsf1b can initiate pro-inflammatory signaling pathways, the host’s pro-inflammatory signaling can limit K. pneumoniae infection. Our findings support the idea that zebrafish tnfrsf1b functions in a manner comparable to its mammalian counterparts, despite sequence divergence. Previous studies in mice have demonstrated that tnfrsf1b signaling promotes regulatory T cell expansion, macrophage polarization, and endothelial repair by activating NF-κB and MAPK signaling cascades. These pathways are also implicated in balancing pro-inflammatory and anti-inflammatory responses during bacterial infections. In zebrafish, the observed increase in macrophages, neutrophils, and inflammatory cytokines in tnfrsf1b mutants may reflect the loss of tnfrsf1b-mediated regulatory signaling, thereby leading to excessive inflammatory responses. Taken together, these results suggest that the protective role of tnfrsf1b against K. pneumoniae infection in zebrafish is at least partly mediated through conserved downstream pathways such as NF-κB and MAPK, which parallels findings from mammalian systems.

One of the advantages of zebrafish as a research model organism is that they can facilitate pharmacological operations because we can dissolve drugs into the culture water, and zebrafish larvae can absorb the drugs through the skin.²⁹ Inhibitors of TNFR can act on both of the receptor proteins.³⁰ In this study, an inhibitor of TNFR was tested to mimic the phenotype observed in the tnfrsf1b knockout. The inhibitor plus K. pneumoniae infection of larvae zebrafish led to a significant increase in teratogenicity and mortality and an increase in immune cell numbers; however, the result is inconsistent in the quantification of immune genes. The main reason may be that TNFR inhibitors not only act on tnfrsf1b but also act on the activity of TNFR1. Research has shown that TNFR1 and tnfrsf1b participate in different immune processes.³¹ TNFR1 can activate the transcription factor NF-κB, mediate apoptosis, and function as a regulator of inflammation.³² tnfrsf1b and TNFR1 form a heterocomplex that mediates the recruitment of two anti-apoptotic proteins, c-IAP1 and c-IAP2, which possess E3 ubiquitin ligase activity.³³ tnfrsf1b has no intracellular death domain; thus, this protein protects neurons from apoptosis by stimulating antioxidative pathways.³⁴ Whether there are other targets for the pharmacological mechanism of action needs further study.

tnfrsf1b can be used as a candidate target to treat K. pneumoniae infection. Injecting the tnfrsf1b-capped mRNA into the tnfrsf1b mutant rescued the phenotype caused by K. pneumoniae and also improved the phenotype of K. pneumoniae infection in WT zebrafish. It is noteworthy that in Figure 5A the deformity rate of tnfrsf1b mutants showed an increasing trend after K. pneumoniae infection, although the difference did not reach statistical significance. In contrast, mortality significantly increased (Figure 5B). This discrepancy may be explained by the fact that larvae with more severe developmental deformities were less tolerant to infectious stress, thereby contributing to the elevated mortality. These findings suggest that tnfrsf1b deficiency may synergize developmental abnormalities and immune dysfunction to exacerbate susceptibility to bacterial infection. Previous research has mainly focused on the development of antibiotics to kill or resist pathogens; however, whether K. pneumoniae can cause disease through internal changes in the body and the role of bacterial elimination is a direction worth studying in the future. Previous studies on tnfrsf1b have found that it can play a role in neurodevelopment and anti-oxidation.³⁴

Recent studies have further elucidated the immunomodulatory role of tnfrsf1b (TNFR2) in bacterial infection contexts. For instance, Liang et al (2022) demonstrated that while TNFR1 signaling promotes inflammatory responses and effector T cell expansion, TNFR2 signaling is distinctly associated with anti-inflammatory activity and tissue homeostasis, underscoring the balancing role of tnfrsf1b in host defense.³⁵ Moreover, Zhang et al (2024) reported that GRN can upregulate TNFR2 expression and promote M2 macrophage polarization, further supporting a role for tnfrsf1b in orchestrating macrophage phenotype toward tissue repair and resolution of inflammation.³⁶ Additionally, in a bacteremia model, Xu et al (2023) found that TNFR2⁺ regulatory T cells are essential for protecting against pulmonary injury, indicating that tnfrsf1b contributes to immune tolerance mechanisms during severe infection.³⁷ Collectively, these findings align with our observations in zebrafish where tnfrsf1b deficiency leads to exaggerated inflammation and immune cell infiltration, suggesting that macrophage polarization and regulatory T cell pathways may be key mechanisms through which tnfrsf1b modulates host responses to K. pneumoniae infection.

Our study demonstrated that tnfrsf1b-deficient zebrafish exhibit exaggerated inflammatory responses and increased mortality upon K. pneumoniae infection, suggesting that tnfrsf1b plays a protective role. However, whether tnfrsf1b is strictly essential for resistance to K. pneumoniae remains an open question. It is possible that tnfrsf1b functions as a modulatory rather than indispensable factor, fine-tuning the balance between pro- and anti-inflammatory signals during infection. Moreover, the interaction between host genetic deficiency and bacterial virulence is complex, and the exacerbated phenotypes observed may reflect a synergistic effect rather than independent contributions. Future work employing double manipulations (eg, tnfrsf1b mutants combined with infection by different virulence strains, or selective pathway inhibition) will be important to dissect whether the effects of tnfrsf1b deficiency and K. pneumoniae infection are additive or synergistic.

It should be noted that R7050 is a pan-TNFR inhibitor that blocks the activity of both TNFR1 and tnfrsf1b. Therefore, the phenotypes observed in R7050-treated zebrafish may not exclusively reflect the inhibition of tnfrsf1b. TNFR1 is known to activate pro-inflammatory signaling pathways such as NF-κB and apoptosis in mammalian systems, and its inhibition could potentially alter the balance between inflammatory activation and resolution. Nevertheless, the fact that tnfrsf1b mutants and R7050-treated zebrafish showed comparable immune dysfunction supports the conclusion that tnfrsf1b plays a major role in the observed phenotypes. We have revised the Discussion to clarify this point and to highlight that future work using more selective inhibitors or genetic approaches will help to dissect the relative contributions of TNFR1 and tnfrsf1b.

While our findings highlight tnfrsf1b as a potential therapeutic target for mitigating immune dysfunction during K. pneumoniae infection, caution must be exercised when considering its translational application. In mammalian models, excessive activation of tnfrsf1b has been linked to immunosuppression and impaired pathogen clearance. Therefore, therapeutic strategies aimed at enhancing tnfrsf1b signaling may reduce damaging inflammation but could simultaneously increase susceptibility to secondary infections. This dual effect underscores the need for carefully balanced approaches that restore immune homeostasis without tipping toward excessive suppression. Future studies should evaluate the safety and long-term consequences of modulating tnfrsf1b activity in the context of bacterial infections.

One limitation of this study is the relatively low amino acid sequence identity between zebrafish tnfrsf1b and its mammalian counterparts (21.8–24.15%). This raises concerns regarding the direct extrapolation of our findings to humans. Although structural analysis suggests that key signaling domains are conserved, the differences in immune system architecture between zebrafish and mammals may result in species-specific variations in tnfrsf1b function. Therefore, while zebrafish provide a powerful vertebrate model for dissecting host–pathogen interactions in vivo, caution is warranted in directly translating these results to human K. pneumoniae infections. Future studies in mammalian models will be important to validate the mechanistic insights obtained from zebrafish.

In conclusion, our study demonstrates that tnfrsf1b deficiency in zebrafish leads to increased susceptibility to K. pneumoniae infection, whereas restoration of tnfrsf1b signaling confers protection. These findings suggest that tnfrsf1b may represent a candidate pathway for further investigation as a potential therapeutic target against hypervirulent K. pneumoniae infection, although validation in mammalian models and clinical settings will be required.