Trypanosomiasis-induced Th17-like immune responses in carp

Abstract

Background: In mammalian vertebrates, the cytokine interleukin (IL)-12 consists of a heterodimer between p35 and p40 subunits whereas interleukin-23 is formed by a heterodimer between p19 and p40 subunits. During an immune response, the balance between IL-12 and IL-23 can depend on the nature of the pathogen associated molecular pattern (PAMP) recognized by, for example TLR2, leading to a preferential production of IL-23. IL-23 production promotes a Th17-mediated immune response characterized by the production of IL-17A/F and several chemokines, important for neutrophil recruitment and activation. For the cold blooded vertebrate common carp, only the IL-12 subunits have been described so far.

Methodology/principal findings: Common carp is the natural host of two protozoan parasites: Trypanoplasma borreli and Trypanosoma carassii. We found that these parasites negatively affect p35 and p40a gene expression in carp. Transfection studies of HEK293 and carp macrophages show that T. carassii-derived PAMPs are agonists of carp TLR2, promoting p19 and p40c gene expression. The two protozoan parasites induce different immune responses as assessed by gene expression and histological studies. During T. carassii infections, in particular, we observed a propensity to induce p19 and p40c gene expression, suggestive of the formation of IL-23. Infections with T. borreli and T. carassii lead to an increase of IFN-γ2 gene expression whereas IL-17A/F2 gene expression was only observed during T. carasssii infections. The moderate increase in the number of splenic macrophages during T. borreli infection contrasts the marked increase in the number of splenic neutrophilic granulocytes during T. carassii infection, along with an increased gene expression of metalloproteinase-9 and chemokines.

Conclusion/significance: This is the first study that provides evidence for a Th17-like immune response in fish in response to infection with a protozoan parasite.

Figure 1. Activation of carp TLR2 by parasite-derived PAMPs in HEK 293 cells.

TLR2WT- and TLR2ΔTIR-transfected HEK 293 cells were stimulated with live T. borreli or T. carassii parasites (Live Tb or Live Tc, 0.5 million parasites per well), with supernatants from 5 million PI-PLC-treated- T. borreli or -T. carassii parasites (GPI Tb or GPI Tc) or left untreated as negative control. MAPK-p38 phosphorylation was analysed by immunoblotting for phospho-p38 (P-p38), while equal loading was confirmed by immunoblotting for β-tubulin (A). Fold change of P-p38 and β-tubulin protein levels in the stimulated cells of TLR2WT- and TLR2ΔTIR-transfected HEK 293 cells are shown relative to the untreated cells. The P-p38 signal intensity of TLR2ΔTIR-transfected HEK 293 cells was below the threshold and therefore non-detectable (N.D.) (B). One experiment representative of three independent experiments is shown.

Figure 2. Kinetics of gene expression of IL-12 family members in head kidney during infection with T. borreli (A) or T. carassii (B).

Carp were injected (i.p.) with a dose of 10000 parasites per fish, PBS-injected individuals served as negative controls (week 0). At indicated time points n = 5 (T. borreli) or n = 4 (T. carassii) animals were sacrificed for organ collection. Parasitaemia was monitored during infection and is shown in the upper plot, at a logarithmic scale. mRNA levels of IL-12 family members are shown relative to the house keeping gene 40S ribosomal protein S11. Data points represent averages + SD of n = 4-5 fish per time point. Symbol (*) represents a significant (P≤0.05) difference compared to non-infected fish.

Figure 3. Kinetics of gene expression of IL-12 family members in re-stimulated head kidney leukocytes of T. borreli- (A) or T. carassii- (B) infected carp.

Carp were injected (i.p.) with PBS or a dose of 10000 parasites per fish. Head kidney leukocytes (HKL) from non-infected (n = 1) or infected fish (n = 3) were isolated at different time points post-infection and re-stimulated with PGN (50 µg/mL) or homologous live parasites (0.25 million parasites per well) for 6 h. mRNA levels of IL-12 family members were normalized against the house keeping gene 40S ribosomal protein S11 and shown as fold change relative to unstimulated head kidney leukocytes. Data points represent averages + SD of n = 3 non-infected fish (taken at the three time points analysed and indicated as week 0) and n = 3 infected fish per time point. Symbol (*) represents a significant (P≤0.05) difference between PGN re-stimulated HKL from infected fish compared to PGN re-stimulated HKL from non-infected fish. Symbol (#) represents a significant (P≤0.05) difference between parasite re-stimulated HKL from infected fish compared to parasite re-stimulated HKL from non-infected fish.

Figure 4. Overexpression of TLR2 in carp macrophages.

TLR2ΔTIR- and TLR2WT-transfected carp macrophages were stimulated with live T. borreli or T. carassii parasites (0.25 million parasites per well) for 6 h, or left untreated as negative control. mRNA levels of IL-12 family members were normalized against the house keeping gene 40S ribosomal protein S11 and are shown as fold change relative to unstimulated macrophages (fold change  = 1). Bars show averages + SD of n = 4 fish. Symbol (*) represents a significant (P≤0.05) difference in gene expression between parasite-stimulated cells macrophages in TLR2WT-transfected- compared to TLR2ΔTIR- transfected carp macrophages.

Figure 5. Macrophage (A) and neutrophilic granulocyte (B) cell populations in spleen of T. borreli- and T. carassii- infected fish.

Carp were injected (i.p.) with a dose of 10000 parasites per fish, PBS-injected individuals served as negative controls. Spleens from non-infected and infected fish were collected at different time points post-injection. Monocytes/macrophages (WCL-15+) and neutrophilic granulocytes (TCL-BE8+) are stained in red. One experiment representative of three independent experiments is shown.

Figure 6. Constitutive gene expression of IL-12 family members in neutrophilic granulocytes from T. carassii- infected fish.

Carp were injected (i.p.) with a dose of 10000 parasites per fish, PBS-injected individuals served as negative controls. Three weeks post-infection, splenocytes from non-infected (n = 2) or T. carassii-infected fish (n = 2) were isolated and neutrophilic granulocytes (TCL-BE8+ cells) sorted to purify by magneting sorting. Each bar represents a single fish.

Figure 7. IFN-γ2 and IL-17A/F2 gene expression during T. borreli- and T. carassii-infection.

Gene expression was measured at different time points post-infection in peripheral blood leukocytes (PBL) (A) or at a fixed time point (3 weeks) in head kidney (HK), mid kidney (MK) and spleen (SP) (B). Carp were injected (i.p.) with a dose of 10000 parasites per fish, PBS-injected individuals served as negative controls. mRNA levels of IFN-γ2 and IL-17A/F2 were normalized against the house keeping gene 40S ribosomal protein S11 and expressed as fold change relative to non-infected fish. Data points represent averages + SD of n = 3 non-infected fish (indicated as week 0) and n = 3 infected fish per time point. Symbol (*) represents a significant (P≤0.05) difference compared to non-infected fish.