Fig 1.
Structural characterization of immunoglobulins in trout nasal mucus.
(A) Fractionation of nasal mucus (~ 0.5 ml) by gel filtration (upper) followed by immunoblot analysis of the fractions with anti-trout IgM-, anti-trout IgD-specific mAbs, and anti-trout IgT-specific pAbs (lower). A280, absorbance at 280 nm. (B) SDS-PAGE of gel-filtration fractions (4–15%) corresponding to eluent (40 μl) at elution volumes of 8.5 ml and 11.5 ml under non-reducing conditions followed by immunoblot analysis with anti-trout IgM-, anti-trout IgD-specific mAbs or anti-trout IgT-specific pAbs. (C and D) Immunoblot and densitometric analysis of the concentration of IgT, IgM and IgD in nasal mucus (40 μl) (C) and serum (0.5 μl) (D) (n = 12 fish). Ratio of IgT to IgM concentration (E) and IgD to IgM concentration (F) in nasal mucus and serum, calculated from the values shown in C and D. Results in figs C-F are expressed as mean and s.e.m. obtained from 12 individual fishes.
Fig 2.
Trout pIgR associates nasal sIgT.
(A) SDS-PAGE under reducing conditions of trout serum and nasal mucus (~ 5 μg each), followed by immunoblot analysis using anti-trout pIgR antibody. (B) Co-immunoprecipitation (CoIP) of nasal mucus with anti-trout IgT antibody, followed by immunoblot analysis under reducing conditions (pIgR detection, upper panels) or non-reducing conditions (IgT detection, lower panels). (C) Immunofluorescence staining for pIgR with IgT in olfactory organ paraffinic sections of rainbow trout. Differential interference contrast images of olfactory organ paraffin sections were stained with anti-trout pIgR (magenta), anti-trout IgT (green) and DAPI for nuclei (blue) (n = 9) (isotype-matched control antibodies for anti-pIgR in S1A Fig). Enlarged sections of the areas outlined showing some pIgR/IgT colocalization (white arrowhead). NC, nasal cavity; OE, olfactory epithelium; LP, lamina propria. Scale bar, 20 μm. Data are representative of at least three different independent experiments.
Fig 3.
Kinetics of immune response and pathological changes in trout olfactory organ following Ich parasites infection.
(A) Heat map illustrates results from quantitative real-time PCR of mRNAs for selected immune markers in parasite-challenged versus control fish measured at 1, 7, 14, 21, 28 and 75 days post infections with Ich parasite in the olfactory organ of rainbow trout (n = 6 fish per group). Color value: log2 (fold change). (B) Relative expression of IgM, IgD and IgT at 1, 7, 28 and 75 days post infection with Ich parasite in olfactory organ of rainbow trout (n = 6 fish per group). (C) Histological examination (haematoxylin & eosin stain; H & E) of the olfactory organ from ich-infected rainbow trout 7, 28, 75 d.p.i and uninfected fish (n = 6 fish per group). Black arrows indicate the width of LP at the tip region (100 μm from the lamellar tip) of the olfactory lammella and red arrows indicate goblet cells. (D) The width of LP at the tip region the olfactory lamella in ich-infected rainbow trout 7, 28, 75 d.p.i and control fish counted from C. (E) The number of goblet cells at the olfactory lamella in ich-infected rainbow trout 7, 28, 75 d.p.i and control fish counted from C. *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 and s.e.m.). Anova, analysis of variance.
Fig 4.
IgT coats Ich parasite located in olfactory organ of infected trout.
Four different microscope images (A-D) of slides immunofluorescence staining of Ich parasites in olfactory organ paraffinic sections from trout infected with Ich after 28 days (n = 6). (A and B) Immunofluorescence stained with Ich (magenta), IgM (red) and IgT (green), nuclei stained with DAPI (blue) (from left to right). (C and D) Immunofluorescence stained with Ich (magenta), IgD (red) and IgT (green) with nuclei stained with DAPI (blue) (from left to right); DIC images showing merged staining (isotype-matched control antibody staining, S3A–S3C Fig). Scale bars, 20 μm. Data are representative of at least three different independent experiments.
Fig 5.
Accumulation of IgT+ B cells in the olfactory organ of trout infected with Ich.
DIC images of immunofluorescence staining on trout nasal paraffinic sections from uninfected fish (A), 28 days infected fish (B) and survivor fish (C), stained for IgT (green) and IgM (red); nuclei are stained with DAPI (blue). (D) Enlarged images of the areas outlined in c are showing some IgT+ B cells possibly secreting IgT (white arrowhead) (isotype-matched control antibody staining, S1B Fig). NC, nasal cavity; OE, olfactory epithelium; LP, lamina propria. Scale bar, 20 μm. Data are representative of at least three different independent experiments (n = 8 per group).
Fig 6.
Increases of IgT+ B cells and IgT concentration in the olfactory organ of trout infected with Ich.
Percentage of IgT+ and IgM+ B cells in NALT (A) and head kidney (B) leukocytes of uninfected control fish, infected fish and survivor fish measured by flow cytometric analysis (n = 12 per group). Concentration of IgT, IgM and IgD in nasal mucus (C) and serum (D) of control, infected and survivor fish (n = 12 per group). *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 and s.e.m.). Anova, analysis of variance.
Fig 7.
Immunoglobulin responses in the nasal mucus and serum from infected and survived trout.
(A) Western blot analysis of IgT-, IgM- and IgD- specific binding to Ich in nasal mucus (dilution 1:2) from infected and survivor fish. (B and C) IgT-, IgM- and IgD- specific binding to Ich in dilutions of nasal mucus from infected (B) and survivor (C) fish, evaluated by densitometric analysis of immunoblots and presented as relative values to those of control fish (n = 8 per group). (D) Western blot analysis of IgT-, IgM- and IgD- specific binding to Ich in serum (dilution 1:10) from infected and survivor fish. (E and F) IgT-, IgM- and IgD- specific binding to Ich in dilutions of serum from infected (E) and survivor (F) fish, evaluated by densitometric analysis of immunoblots and presented as relative values to those of control fish (n = 8 per group). *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 and s.e.m.).
Fig 8.
Proliferative responses of IgT+ and IgM+ B cells in the olfactory organ of survived trout.
Immunofluorescence analysis of EdU incorporation by IgT+ or IgM+ B cells in olfactory organ of control (A) and survivor fish (B). Nasal paraffin sections were stained for EdU (magenta), trout IgT (green), trout IgM (red) and nuclei (blue) detection (n = 8 fish per group). NC, nasal cavity; OE, olfactory epithelium; LP, lamina propria. Scale bars, 20 μm. (C) Percentage of EdU+ cells from total nasal cell in control or survivor fish counted from Fig 7A and 7B (n = 8). (D) Percentage of EdU+ cells from the total IgT+ or IgM+ B cells populations in olfactory organ of control and survivor fish counted from A and B. Data in A and B are representative of at least three independent experiments (mean and s.e.m.). Statistical analysis was performed by unpaired Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001.
Fig 9.
Local IgT-, IgM- and IgD-specific responses in olfactory organ explants of survivor fish.
The olfactory organ, head kidney and spleen explants (~ 20 mg each) from control and survivor fish were cultured in medium (400 μl) for 7 days. Immunoblot analysis of IgT-, IgM- and IgD-specific binding to Ich in the culture medium of olfactory organ (A), head kidney (B) and spleen (C) (dilution 1:2) from control and survivor fish. (D-F) IgT-, IgM- and IgD-specific binding to Ich in dilutions of culture medium from olfactory organ (D), head kidney (E) and spleen (F) from control and survivor fish, measured by densitometric analysis of immunoblots and presented as relative values to those of control fish (n = 6–8 per group). *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 and s.e.m.).
Fig 10.
Proposed model of local IgT and IgT+ B cell induction in the olfactory organ.
Model images represent a fish (A) and enlarged sections of the areas outlined in a showing the olfactory organ (B). (C) Induction of local IgT responses in the trout olfactory organ based on our findings. When Ich parasite invaded the nasal mucosa, Ich antigen (Ag) are taken up by antigen-presenting cells (APC) and presented to naïve CD4-T cells. Ag-specific CD4-T cells then produced cytokines to activate B cells. Activated B cells start proliferating in olfactory organ and may differentiation to plasma cells to locally produce Ich-specific IgT, which will be transported by pIgR into nasal mucus where can specific binding to the Ich parasite. Alternatively, some IgT+ plasma cells may differentiate into memory IgT+ B cells. When Ich parasite infection happened again in olfactory organ, memory IgT+ B cells directly proliferate and differentiate into plasma cells and produce larger amounts of specific-IgT to binding Ich. The trout olfactory organ showing the mucosal tip area with goblet cells and the lateral neuroepithelium. NC, nasal cavity; OM, olfactory mucus; OE, olfactory epithelium; BL, basal layer; LP, lamina propria.