Response of immunoglobulin M in gut mucosal immunity of common carp (Cyprinus carpio) infected with Aeromonas hydrophila

Abstract

Immunoglobulin (Ig) M is an important immune effector that protects organisms from a wide variety of pathogens. However, little is known about the immune response of gut mucosal IgM during bacterial invasion. Here, we generated polyclonal antibodies against common carp IgM and developed a model of carp infection with Aeromonas hydrophila via intraperitoneal injection. Our findings indicated that both innate and adaptive immune responses were effectively elicited after A. hydrophila infection. Upon bacterial infection, IgM⁺ B cells were strongly induced in the gut and head kidney, and bacteria-specific IgM responses were detected in high levels both in the gut mucus and serum. Moreover, our results suggested that IgM responses may vary in different infection strategies. Overall, our findings revealed that the infected common carp exhibited high resistance to this representative enteropathogenic bacterium upon reinfection, suggesting that IgM plays a key role in the defense mechanisms of the gut against bacterial invasion. Significantly, the second injection of A. hydrophila induces strong local mucosal immunity in the gut, which is essential for protection against intestinal pathogens, providing reasonable insights for vaccine preparation.

Keywords: Aeromonas hydrophila; B cells; IgM; common carp; mucosal immune response.

Figure 1

The common carp IgM polyclonal antibody (pAb) production and characterization. (A) Structural schematic of pET-32a-IgM recombinant plasmids. (B) Coomassie blue staining of recombinant protein of IgM resolved by SDS-PAGE. Red arrowheads indicate the recombinant IgM. (C) Western blot analysis with pAb to common carp serum. Left margin, molecular size in kilodaltons (kDa). MK, marker; NR, nonreducing conditions; RE, reducing conditions. (D) Immunofluorescence staining of IgM⁺ B cells in different tissues of common carp. Tissue paraffinic sections were stained with rabbit anti-carp IgM (green) and nuclei were stained with DAPI (blue) (isotype-matched control antibody staining, Figure S1A ). White arrowheads point to cells stained for IgM. White dotted lines outline the border. Scale bar, 20 μm. EP, epidermis; DE, dermis; PL, primary lamella; SL, secondary lamella; LU, lumen; LP, lamina propria; BC, buccal cavity; BE, buccal epithelium; PC, pharyngeal cavity; PE, pharyngeal epithelium; NC, nasal cavity; OE, olfactory epithelium.

Figure 2

Scheme of the infection strategy with A. hydrophila via intraperitoneal injection. (A) Fish were injected with A. hydrophila and sacrificed at 0.5, 1, 2, 4, 7, 14, and 28 DPI for sample collection, while fish were injected with PBS as control. (B) The clinical observation following challenge with A. hydrophila. (C–E) Histological examination by H&E staining of foregut (C), midgut (D), and hindgut (E) from control and infected fish (n = 6). Scale bars, 50 μm. (F–H) The length-width ratio of foregut (F), midgut (G), and hindgut (H) intestinal folds in these groups of fish from (C–E) (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA with Bonferroni correction). Data are representative of at least three independent experiments (mean ± SEM).

Figure 3

Immune response in gut, spleen, and head kidney tissues of common carp following A. hydrophila infection. (A–E) Heat map illustrates results from qPCR of mRNAs for selected immune-related genes in bacteria-challenged fish vs. control group measured at 0.5, 1, 2, 4, 7, 14, and 28 DPI following with A. hydrophila in the foregut (A), midgut (B), hindgut (C), spleen (D), and head kidney (E) organs of common carp (n = 6). Color value: log₂ (fold change). Data are representative of at least three different independent experiments.

Figure 4

Scheme of the reinfection strategy and accumulation of IgM⁺ B cells of common carp infected with A. hydrophila. (A) Scheme of the rechallenge strategy with A. hydrophila via intraperitoneal injection. Fish were injected with A. hydrophila at 0 days and reinfected at 28 DPI with the same bacterial dose, and the resulting surviving fish were sacrificed at 28 and 35 days after the primary infection (28 DPI surviving group, [28DPI-S]) and 35 (35 DPI surviving group, [35DPI-S]). The three groups of fish include Control (PBS + PBS), Con+Cha (PBS + A. hydrophila), and Infe+Cha (A. hydrophila + A. hydrophila). (B–F) Representative differential interference contrast (DIC) images of immunofluorescence staining on common carp foregut (B), midgut (C), hindgut (D), spleen (E), and head kidney (F) paraffin-sections from uninfected (Control) fish, 28DPI-S, and 35DPI-S fish, stained for IgM (green) (Isotype-matched control antibody staining is shown in Figure S1A ); nuclei (blue) were stained with DAPI (blue). Scale bars, 20 μm. (G–K) The number of IgM⁺ B cells of Control, 28DPI-S, and 35DPI-S fish counted from (B–F) (n = 6). ns, not significant, *P < 0.05, **P < 0.01, and ***P < 0.001 (unpaired Student’s t-test). Data are representative of at least three independent experiments (mean ± SEM).

Figure 5

IgM responses in the gut mucus and serum from 28DPI-S and 35DPI-S fish. (A–D) The concentration of IgM protein in foregut mucus (A), midgut mucus (B), hindgut mucus (C), and serum (D) of Control, 28DPI-S, and 35DPI-S fish (n = 6). (E–H) Western blot analysis of IgM-specific binding to A. hydrophila in foregut mucus (E), midgut mucus (F), hindgut mucus (G), and serum (H) (mucus dilution 1:2; serum dilution 1:10) from 28DPI-S and 35DPI-S fish. (I–L) IgM-specific binding to A. hydrophila in dilutions of foregut mucus (I), midgut mucus (J), hindgut mucus (K), and serum (L) from 28DPI-S and 35DPI-S fish, evaluated by densitometric analysis of immunoblots and presented as relative values to those of control fish (n = 6). ns, not significant, *P < 0.05, **P < 0.01, and ***P < 0.001 (unpaired Student’s t-test). Data are representative of at least three independent experiments (mean ± SEM).

Figure 6

Histological examination, bacterial loads, and survival rates of common carp after rechallenging with A. hydrophila. (A–C) Histological examination by H&E staining of foregut (A), midgut (B), and hindgut (C) from Control (Con-7d), Con+Cha (Infe-7d), and Infe+Cha (Reinfe-7d) groups of fish. (D) The length-width ratio of foregut, midgut, and hindgut intestinal folds from (A–C). (E) The expression levels of A. hydrophila in control, infected-7d, and reinfected-7d fish were measured in the gut of common carp (n = 6). (F) Cumulative survival of control, infected, and reinfected fish. Statistical differences were evaluated by log-rank (Mantel-Cox) test. ns, not significant, *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA with Bonferroni correction). Data are representative of at least three independent experiments (mean ± SEM).