Lack of endogenous IL-10 enhances production of proinflammatory cytokines and leads to Brucella abortus clearance in mice

Abstract

IL-10 is a cytokine that regulates the balance between pathogen clearance and immunopathology. Brucella abortus is an intracellular bacterium that causes chronic disease in humans and domestic animals. Here we evaluated the contribution of IL-10 in host immune response and pathology during B. abortus infection. To assess the role of IL-10 in vivo, IL-10 knockout (KO) or 129 Sv/Ev (wild-type) mice were infected with B. abortus and the number of viable bacteria from the spleen was determined at 1, 2, 3, 6 and 14-weeks postinfection. IL-10 KO mice showed reduced bacterial loads in the spleen when compared to wild-type mice during all time points studied. Additionally, at 14-weeks postinfection IL-10 KO mice had totally cleared the infection. This clearance was preceded by an enhanced IFN-γ, TNF-α and IL-17 responses in both the serum and the spleen of IL-10 KO mice. Additionally, dendritic cells from infected IL-10 KO mice produced elevated levels of IL-12 and TNF-α compared to wild-type animals. Histopathology analysis was performed and both KO and wild-type mice developed multifocal granulomas and necrosis in the liver. However, at six-weeks postinfection reduced numbers of granulomas was detected in IL-10 KO mice compared to wild-type animals. This reduced liver pathology at later stage of infection was accompanied by increased numbers of CD4+CD25+foxp3+ T cells and expression of TGF-β in IL-10 KO splenocytes. Taken together, our findings demonstrate that IL-10 modulates the proinflammatory immune response to B. abortus infection and the lack of IL-10 increases resistance to Brucella infection.

Figure 1. Brucella induces IL-10 production in BMDCs and spleen cells in 129 Sv/Ev mice.

(A) Bone marrow cells from wild-type or IL-10 KO mice were differentiated in dendritic cells (BMDC) and infected with B. abortus (MOI 1∶100) or stimulated with E. coli LPS (1 µg/ml). IL-10 was measured by ELISA at 24 hours after antigen stimulation. (B) Spleen cells from B. abortus infected mice at 1, 2, 3 or 6 weeks postinfection were cultured with 10² bacteria/cell, ConA (5 µg/ml) or medium alone for 72 hours. Supernatants were harvested for measuring IL-10 by ELISA. The level of IL-10 at week 0 was below 30 pg/ml. Error bars represent the mean ±SD. Similar results were obtained in four-independent experiments. Statistically significant differences of IL-10 levels from IL-10 KO mice compared to wild-type are denoted by an asterisk (p<0.05). Differences of IL-10 levels from wild-type mice stimulated with Brucella or the positive control compared to medium alone are denoted by & (p<0.05). (C) Flow cytometry analysis of CD4+, CD8+, CD19+, CD11c+ and CD11b+/F4/80+ cells producing IL-10 was carried out in splenocytes from Brucella-infected mice at one-week post-infection. Statistically significant differences of IL-10 levels from IL-10 KO mice compared to wild-type are denoted by an asterisk (p<0.05).

Figure 2. IL-10 is required to control proinflammatory cytokine production by BMDCs infected with B. abortus.

Bone marrow cells from wild-type or IL-10 KO mice were differentiated in dendritic cells and infected with B. abortus (MOI, 1∶100) or stimulated with E. coli LPS (1 µg/ml). Supernatants were harvested for measuring IL-12 (A) or TNF-α (B) at 24 hours after infection/stimulation by ELISA. Error bars represent the mean ±SD. Similar results were obtained in four-independent experiments. Statistically significant differences relative to medium (nonstimulated cells) are denoted by & (p<0.05) and differences from IL-10 KO mice compared to wild-type are indicated by an asterisk (p<0.05).

Figure 3. Bacterial burden and IL-12, TNF-α, IFN-γ and IL-17 production in Brucella-infected IL-10 KO mice.

Five wild-type or IL-10 KO mice were infected i.p. with a dose of 10⁶ CFU of B. abortus for bacterial burden analysis or a dose of 10⁹ CFU of B. abortus for serum cytokine analysis. (A) Spleens were harvested at 1, 2, 3, 6 or 14 weeks post-infection, and the number of CFU in disrupted tissue was determined by 10-fold serial dilution and plating. Statistically significant differences in CFU of IL-10 KO compared to wild-type mice are denoted by an asterisk (p<0.05). IL-12 (B), IFN-γ (C), IL-17 (D) or TNF-α (E) production in mice sera were measured by ELISA at 24 hours postinfection. Error bars represent the mean ±SD. Similar results were obtained in four-independent experiments. Statistically significant differences of cytokine levels from IL-10 KO mice compared to wild-type are denoted by an asterisk (p<0.05) and statistically significant differences to time 0 hour are denoted by & (p<0.05).

Figure 4. Endogenous IL-10 is required to control proinflammatory cytokine production by splenocytes infected with B. abortus.

Spleen cells from wild-type or IL-10 KO Brucella-infected mice at 1, 2, 3 or 6 weeks postinfection were stimulated with B. abortus (MOI 1∶100) or medium alone as a negative control. Splenocyte supernatants were harvested for measuring IFN-γ (A), TNF-α (B) or IL-17 (C) levels by ELISA. Data are expressed the mean ±SD for five animals per group. The level of IFN-γ and TNF-α at week 0 was below 30 pg/ml and for IL-17 was undetectable. These results are representative of four independent experiments. Statistically significant differences relative to medium alone are denoted by & and differences of IL-10 KO compared to wild-type mice are indicated by an asterisk (p<0.05).

Figure 5. Morphometric analysis, histopathology and immunohistochemistry of hepatic tissue of B. abortus infected IL-10 KO mice.

(A) Columns indicate volumetric proportions of tissue components. The number of portal space, central lobular vein, granuloma, necrosis and parenchyma were evaluated and transformed in percentage at 1, 2 or 6 weeks postinfection. Statistically significant differences relative to non-infected group (NI) are represented by an asterisk (p<0.05). Differences relative to granuloma number from IL-10 KO mice compared to wild-type mice at six-week postinfection are indicated by #. Similar results were obtained in two-independent experiments. (B-F) Representative of hematoxylin- and-eosin-stained sections of hepatic tissue from wild-type mice uninfected (B) or infected at one- (C), two- (D), three- (E) or six-weeks (F). (H-L) Representative of hematoxylin- and eosin-stained sections of hepatic tissue from IL-10 KO mice uninfected (H) or infected at one- (I), two- (J), three- (K) or six-weeks (L). Immunohistochemistry sections of hepatic tissue from wild-type (G) and IL-10 KO (M) mice containing the B. abortus inside the granuloma. The arrows indicate the B. abortus within the granuloma. Scale bars: 20 µm.

Figure 6. Increased percentage of CD4⁺CD25⁺Foxp3⁺ T cells and TGF-β expression in IL-10 KO mice infected with B. abortus.

(A) Flow cytometry analysis of CD4⁺CD25⁺Foxp3⁺ T cells was carried out in splenocytes from infected (I) or non-infected (NI) mice at 1, 2, 3 or 6 weeks post-infection. Results are expressed as arbitrary units from relative percentage of I/NI CD4⁺CD25⁺Foxp3⁺ T cells encountered in the spleens at indicated time points. The ratio between the percentage of CD4⁺CD25⁺Foxp3⁺ T cells/spleen in I/NI animals was used because of the variation observed on the percentage of these cells in NI mice at different time intervals tested due to the age difference during the duration of the experiment. The experiments were conduct in triplicate and three independent experiments were performed with similar results. Error bars represent the mean ±SD. Statistically significant differences between wild-type and IL-10 KO mice are denoted by an asterisk (p<0.05). (B) Splenocytes from B. abortus infected mice at 1, 2, 3 or 6 weeks postinfection were isolated and total RNA was harvested and mRNA levels of TGF-β1 were determined by real-time RT-PCR and normalized to β-actin. Error bars represent the mean ±SD. Similar results were obtained in three-independent experiments. Statistically significant differences of TGF-β1 expression from IL-10 KO mice compared to wild-type are denoted by an asterisk (p<0.05) and differences of TGF-β expression from infected mice compared to uninfected control (0 week) are denoted by & (p<0.05). (C) splenocyte culture supernatants were harvested for measuring TGF-β1 levels by ELISA. Data are expressed the mean ±SD for five animals per group. Error bars represent the mean ±SD. Statistically significant differences between wild-type and IL-10 KO mice are denoted by an asterisk (p<0.05).