Table_2_miR-181a-5p Regulates TNF-α and miR-21a-5p Influences Gualynate-Binding Protein 5 and IL-10 Expression in Macrophages Affecting Host Control of Brucella abortus Infection.PDF

<p>Brucella abortus is a Gram-negative intracellular bacterium that causes a worldwide zoonosis termed brucellosis, which is characterized as a debilitating infection with serious clinical manifestations leading to severe complications. In spite of great advances in studies involving host–B. abortus interactions, there are many gaps related to B. abortus modulation of the host immune response through regulatory mechanisms. Here, we deep sequenced small RNAs from bone marrow-derived macrophages infected with B. abortus, identifying 69 microRNAs (miRNAs) that were differentially expressed during infection. We further validated the expression of four upregulated and five downregulated miRNAs during infection in vitro that displayed the same profile in spleens from infected mice at 1, 3, or 6 days post-infection. Among these miRNAs, mmu-miR-181a-5p (upregulated) or mmu-miR-21a-5p (downregulated) were selected for further analysis. First, we determined that changes in the expression of both miRNAs induced by infection were dependent on the adaptor molecule MyD88. Furthermore, evaluating putative targets of mmu-miR-181a-5p, we demonstrated this miRNA negatively regulates TNF-α expression following Brucella infection. By contrast, miR-21a-5p targets included a negative regulator of IL-10, programmed cell death protein 4, and several guanylate-binding proteins (GBPs). As a result, during infection, miR-21a-5p led to upregulation of IL-10 expression and downregulation of GBP5 in macrophages infected with Brucella. Since GBP5 and IL-10 are important molecules involved in host control of Brucella infection, we decided to investigate the role of mmu-miR-21a-5p in bacterial replication in macrophages. We observed that treating macrophages with a mmu-miR-21a-5p mimic enhanced bacterial growth, whereas transfection of its inhibitor reduced Brucella load in macrophages. Taken together, the results indicate that downregulation of mmu-miR-21a-5p induced by infection increases GBP5 levels and decreases IL-10 expression thus contributing to bacterial control in host cells.</p

Table_1_miR-181a-5p Regulates TNF-α and miR-21a-5p Influences Gualynate-Binding Protein 5 and IL-10 Expression in Macrophages Affecting Host Control of Brucella abortus Infection.PDF

<p>Brucella abortus is a Gram-negative intracellular bacterium that causes a worldwide zoonosis termed brucellosis, which is characterized as a debilitating infection with serious clinical manifestations leading to severe complications. In spite of great advances in studies involving host–B. abortus interactions, there are many gaps related to B. abortus modulation of the host immune response through regulatory mechanisms. Here, we deep sequenced small RNAs from bone marrow-derived macrophages infected with B. abortus, identifying 69 microRNAs (miRNAs) that were differentially expressed during infection. We further validated the expression of four upregulated and five downregulated miRNAs during infection in vitro that displayed the same profile in spleens from infected mice at 1, 3, or 6 days post-infection. Among these miRNAs, mmu-miR-181a-5p (upregulated) or mmu-miR-21a-5p (downregulated) were selected for further analysis. First, we determined that changes in the expression of both miRNAs induced by infection were dependent on the adaptor molecule MyD88. Furthermore, evaluating putative targets of mmu-miR-181a-5p, we demonstrated this miRNA negatively regulates TNF-α expression following Brucella infection. By contrast, miR-21a-5p targets included a negative regulator of IL-10, programmed cell death protein 4, and several guanylate-binding proteins (GBPs). As a result, during infection, miR-21a-5p led to upregulation of IL-10 expression and downregulation of GBP5 in macrophages infected with Brucella. Since GBP5 and IL-10 are important molecules involved in host control of Brucella infection, we decided to investigate the role of mmu-miR-21a-5p in bacterial replication in macrophages. We observed that treating macrophages with a mmu-miR-21a-5p mimic enhanced bacterial growth, whereas transfection of its inhibitor reduced Brucella load in macrophages. Taken together, the results indicate that downregulation of mmu-miR-21a-5p induced by infection increases GBP5 levels and decreases IL-10 expression thus contributing to bacterial control in host cells.</p

Table_6_miR-181a-5p Regulates TNF-α and miR-21a-5p Influences Gualynate-Binding Protein 5 and IL-10 Expression in Macrophages Affecting Host Control of Brucella abortus Infection.PDF

<p>Brucella abortus is a Gram-negative intracellular bacterium that causes a worldwide zoonosis termed brucellosis, which is characterized as a debilitating infection with serious clinical manifestations leading to severe complications. In spite of great advances in studies involving host–B. abortus interactions, there are many gaps related to B. abortus modulation of the host immune response through regulatory mechanisms. Here, we deep sequenced small RNAs from bone marrow-derived macrophages infected with B. abortus, identifying 69 microRNAs (miRNAs) that were differentially expressed during infection. We further validated the expression of four upregulated and five downregulated miRNAs during infection in vitro that displayed the same profile in spleens from infected mice at 1, 3, or 6 days post-infection. Among these miRNAs, mmu-miR-181a-5p (upregulated) or mmu-miR-21a-5p (downregulated) were selected for further analysis. First, we determined that changes in the expression of both miRNAs induced by infection were dependent on the adaptor molecule MyD88. Furthermore, evaluating putative targets of mmu-miR-181a-5p, we demonstrated this miRNA negatively regulates TNF-α expression following Brucella infection. By contrast, miR-21a-5p targets included a negative regulator of IL-10, programmed cell death protein 4, and several guanylate-binding proteins (GBPs). As a result, during infection, miR-21a-5p led to upregulation of IL-10 expression and downregulation of GBP5 in macrophages infected with Brucella. Since GBP5 and IL-10 are important molecules involved in host control of Brucella infection, we decided to investigate the role of mmu-miR-21a-5p in bacterial replication in macrophages. We observed that treating macrophages with a mmu-miR-21a-5p mimic enhanced bacterial growth, whereas transfection of its inhibitor reduced Brucella load in macrophages. Taken together, the results indicate that downregulation of mmu-miR-21a-5p induced by infection increases GBP5 levels and decreases IL-10 expression thus contributing to bacterial control in host cells.</p

Table_4_miR-181a-5p Regulates TNF-α and miR-21a-5p Influences Gualynate-Binding Protein 5 and IL-10 Expression in Macrophages Affecting Host Control of Brucella abortus Infection.PDF

<p>Brucella abortus is a Gram-negative intracellular bacterium that causes a worldwide zoonosis termed brucellosis, which is characterized as a debilitating infection with serious clinical manifestations leading to severe complications. In spite of great advances in studies involving host–B. abortus interactions, there are many gaps related to B. abortus modulation of the host immune response through regulatory mechanisms. Here, we deep sequenced small RNAs from bone marrow-derived macrophages infected with B. abortus, identifying 69 microRNAs (miRNAs) that were differentially expressed during infection. We further validated the expression of four upregulated and five downregulated miRNAs during infection in vitro that displayed the same profile in spleens from infected mice at 1, 3, or 6 days post-infection. Among these miRNAs, mmu-miR-181a-5p (upregulated) or mmu-miR-21a-5p (downregulated) were selected for further analysis. First, we determined that changes in the expression of both miRNAs induced by infection were dependent on the adaptor molecule MyD88. Furthermore, evaluating putative targets of mmu-miR-181a-5p, we demonstrated this miRNA negatively regulates TNF-α expression following Brucella infection. By contrast, miR-21a-5p targets included a negative regulator of IL-10, programmed cell death protein 4, and several guanylate-binding proteins (GBPs). As a result, during infection, miR-21a-5p led to upregulation of IL-10 expression and downregulation of GBP5 in macrophages infected with Brucella. Since GBP5 and IL-10 are important molecules involved in host control of Brucella infection, we decided to investigate the role of mmu-miR-21a-5p in bacterial replication in macrophages. We observed that treating macrophages with a mmu-miR-21a-5p mimic enhanced bacterial growth, whereas transfection of its inhibitor reduced Brucella load in macrophages. Taken together, the results indicate that downregulation of mmu-miR-21a-5p induced by infection increases GBP5 levels and decreases IL-10 expression thus contributing to bacterial control in host cells.</p

Table_5_miR-181a-5p Regulates TNF-α and miR-21a-5p Influences Gualynate-Binding Protein 5 and IL-10 Expression in Macrophages Affecting Host Control of Brucella abortus Infection.PDF

<p>Brucella abortus is a Gram-negative intracellular bacterium that causes a worldwide zoonosis termed brucellosis, which is characterized as a debilitating infection with serious clinical manifestations leading to severe complications. In spite of great advances in studies involving host–B. abortus interactions, there are many gaps related to B. abortus modulation of the host immune response through regulatory mechanisms. Here, we deep sequenced small RNAs from bone marrow-derived macrophages infected with B. abortus, identifying 69 microRNAs (miRNAs) that were differentially expressed during infection. We further validated the expression of four upregulated and five downregulated miRNAs during infection in vitro that displayed the same profile in spleens from infected mice at 1, 3, or 6 days post-infection. Among these miRNAs, mmu-miR-181a-5p (upregulated) or mmu-miR-21a-5p (downregulated) were selected for further analysis. First, we determined that changes in the expression of both miRNAs induced by infection were dependent on the adaptor molecule MyD88. Furthermore, evaluating putative targets of mmu-miR-181a-5p, we demonstrated this miRNA negatively regulates TNF-α expression following Brucella infection. By contrast, miR-21a-5p targets included a negative regulator of IL-10, programmed cell death protein 4, and several guanylate-binding proteins (GBPs). As a result, during infection, miR-21a-5p led to upregulation of IL-10 expression and downregulation of GBP5 in macrophages infected with Brucella. Since GBP5 and IL-10 are important molecules involved in host control of Brucella infection, we decided to investigate the role of mmu-miR-21a-5p in bacterial replication in macrophages. We observed that treating macrophages with a mmu-miR-21a-5p mimic enhanced bacterial growth, whereas transfection of its inhibitor reduced Brucella load in macrophages. Taken together, the results indicate that downregulation of mmu-miR-21a-5p induced by infection increases GBP5 levels and decreases IL-10 expression thus contributing to bacterial control in host cells.</p

Bacterial burden and IL-12, TNF-α, IFN-γ and IL-17 production in <i>Brucella</i>-infected IL-10 KO mice.

<p>Five wild-type or IL-10 KO mice were infected i.p. with a dose of 10⁶ CFU of <i>B. abortus</i> for bacterial burden analysis or a dose of 10⁹ CFU of <i>B. abortus</i> 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).</p

Mean percentages ± SD of CD4⁺, CD8⁺ or CD19⁺ cells producing IFN-γ or IL-17 in 129 Sv/Ev or IL-10 KO splenocytes at one week post-infection with <i>Brucella abortus</i>.

a<p>Statistically significant compared to 129 Sv/Ev mice.</p

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

<p>(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 <i>B. abortus</i> infected mice at 1, 2, 3 or 6 weeks postinfection were isolated and total RNA was harvested and mRNA levels of <i>TGF-β1</i> were determined by real-time RT-PCR and normalized to <i>β-actin</i>. Error bars represent the mean ±SD. Similar results were obtained in three-independent experiments. Statistically significant differences of <i>TGF-β1</i> expression from IL-10 KO mice compared to wild-type are denoted by an asterisk (p<0.05) and differences of <i>TGF-β</i> 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).</p

<i>Brucella</i> induces IL-10 production in BMDCs and spleen cells in 129 Sv/Ev mice.

<p>(A) Bone marrow cells from wild-type or IL-10 KO mice were differentiated in dendritic cells (BMDC) and infected with <i>B. abortus</i> (MOI 1∶100) or stimulated with <i>E. coli</i> LPS (1 µg/ml). IL-10 was measured by ELISA at 24 hours after antigen stimulation. (B) Spleen cells from <i>B. abortus</i> 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 <i>Brucella</i> 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 <i>Brucella</i>-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).</p

Morphometric analysis, histopathology and immunohistochemistry of hepatic tissue of <i>B. abortus</i> infected IL-10 KO mice.

<p>(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<i><</i>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 <i>B. abortus</i> inside the granuloma. The arrows indicate the <i>B. abortus</i> within the granuloma. Scale bars: 20 µm.</p