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Systemic Expression of Notch Ligand Delta-Like 4 during Mycobacterial Infection Alters the T Cell Immune Response

View Article: PubMed Central - PubMed

ABSTRACT

The Notch ligand delta-like 4 (DLL4) is known to fine-tune the CD4+ T cell cytokine response. DLL4 is expressed on the surface of antigen-presenting cells (APCs) in a MyD88-dependent manner. We found that DLL4 expression was upregulated on bone marrow progenitor cells and APCs in mice infected with BCG Mycobacterium. Transfer of DLL4+ progenitor cells from infected hosts resulted in an increase DLL4+ myeloid cells in the spleen, indicating that expression of the dll4 gene is propagated throughout hematopoiesis. We also found an increase in DLL4+ monocytes from individuals who were infected with Mycobacterium tuberculosis. In latent individuals, DLL4 expression correlated with increased cytokine production from T cells in response to PPD stimulation. Finally, antibody blockade of DLL4 reduced T cell cytokine production from naïve T cells stimulated with antigen. These results demonstrate that the Notch ligand DLL4 can influence T cell cytokine production in both humans and mice, and further reveal that expression of DLL4 is upregulated on early hematopoietic progenitors in response to chronic mycobacterial infection. These data suggest that widespread DLL4 expression may occur as a result of mycobacterial infection, and that this expression may alter CD4+ T cell responses to both previously encountered and novel antigens.

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Delta-like 4 expression correlates with cytokine production from T cells during latent infection. (A) Linear regression analysis of CD4+ T cell IFNγ production, as measured by intracellular cytokine staining, compared to the percent of CD14+ monocytes expressing DLL4 in individuals diagnosed with latent Mtb infection. Similar results were obtained with IL-2 and TNFα production (Figure S4 in Supplementary Material). (B) Same comparison as (A) in individuals with active infection. (C) Production of IFNγ and IL-17 from murine OT-II cells cocultured with OVA peptide and splenic CD11c+CD11b+ FACS sorted from the spleens of naïve mice or mice infected for 5 weeks with BCG in the presence of a polyclonal antibody specific to DLL4 or IgG control antibody. Cells were cultured for 72 h in the presence of 10 μg/mL anti-DLL4 or control antibody. One-way ANOVA analysis indicated DLL4 was a significant factor in determining cytokine output from T cells. For IFNγ, the p value was <0.0001, and for IL-17 the p value was 0.02.
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Figure 4: Delta-like 4 expression correlates with cytokine production from T cells during latent infection. (A) Linear regression analysis of CD4+ T cell IFNγ production, as measured by intracellular cytokine staining, compared to the percent of CD14+ monocytes expressing DLL4 in individuals diagnosed with latent Mtb infection. Similar results were obtained with IL-2 and TNFα production (Figure S4 in Supplementary Material). (B) Same comparison as (A) in individuals with active infection. (C) Production of IFNγ and IL-17 from murine OT-II cells cocultured with OVA peptide and splenic CD11c+CD11b+ FACS sorted from the spleens of naïve mice or mice infected for 5 weeks with BCG in the presence of a polyclonal antibody specific to DLL4 or IgG control antibody. Cells were cultured for 72 h in the presence of 10 μg/mL anti-DLL4 or control antibody. One-way ANOVA analysis indicated DLL4 was a significant factor in determining cytokine output from T cells. For IFNγ, the p value was <0.0001, and for IL-17 the p value was 0.02.

Mentions: Notch ligand expression on APCs has been shown to alter the host immune response to several pathogens (35–39). For example, we have previously demonstrated that T cell-derived IL-17 production is increased as a result of Dll4 expression in a model of mycobacterial infection (31). To determine if DLL4 expression altered cytokine production from T cells in humans infected with M. tuberculosis, we assessed the CD4 T cell production of TNFα, IFNγ, and IL-2 in response to characterized mycobacterial antigens including ESAT-6, TB10.4, Ag85A, CFP-10, and PPD by intracellular cytokine staining in both actively and latently infected individuals. We also assessed the production of these same cytokines in response to the superantigen Staphylococcal Enterotoxin B (SEB) as a positive control. We then performed PCA using these data, including the percentage of DLL4+ monocytes as a factor to determine if DLL4 expression in monocytes was correlated with cytokine production from T cells in latently and actively infected individuals. PCA analysis is routinely used to reduce the number of dimensions in a data set by determining if specific variables track together as a component (40). Our analysis of latently infected individuals resulted in two components each with an Eigenvalue ≥4. The values in Table 1 state that the correlation between each variable and the two discovered principal components on a scale of 1 to −1, with 1 being a 100% correlation. Values of less than 0.3 are considered non-significant. Based on this analysis, we determined that DLL4 expression was correlated with cytokine production from CD4 T cells as a result of stimulation with specific TB antigens including ESAT-6 and PPD in an antigen-specific component. The second component contained all of the cytokine data related to SEB stimulation and was not correlated with DLL4 expression on monocytes. Interestingly, CFP-10 cytokine secretion was associated with the SEB component, suggesting a different pattern of cytokine secretion than that induced by the other antigens. Based on our component matrix (Table S1 in Supplementary Material), which correlates all variables in the data set, we determined that DLL4 expression was most closely correlated with the cytokine response following stimulation with PPD (Table S1 in Supplementary Material). Table S2 in Supplementary Material states that the component containing SEB cytokine stimulation was not related to the component containing DLL4 expression and cytokine production in response to TB antigens. Performing dimension reduction analysis on individuals diagnosed with active TB disease did not result in a component containing DLL4 expression. This analysis informed us that DLL4 expression on monocytes was associated with T cell cytokine production in latently infected individuals. We then performed linear regression analysis to determine if there was a direct correlation between cytokine production and DLL4 expression in both latent infection and active TB disease. Our results indicate an r2 value of 0.54 when correlating the percent of CD4+ T cells producing IFNγ and the percentage of monocytes expressing DLL4 in response to PPD. There was no correlation between DLL4 expression and cytokine production in response to SEB (Figure 4A). There was no correlation between cytokine production and DLL4 expression on monocytes in patients with active TB disease (Figure 4B). There was also no correlation between CD8+ T cell cytokine production and DLL4 expression (Table S3 in Supplementary Material). Consistent with previous data, there was also reduced frequency of cytokine-producing T cells in patients with active TB (33).


Systemic Expression of Notch Ligand Delta-Like 4 during Mycobacterial Infection Alters the T Cell Immune Response
Delta-like 4 expression correlates with cytokine production from T cells during latent infection. (A) Linear regression analysis of CD4+ T cell IFNγ production, as measured by intracellular cytokine staining, compared to the percent of CD14+ monocytes expressing DLL4 in individuals diagnosed with latent Mtb infection. Similar results were obtained with IL-2 and TNFα production (Figure S4 in Supplementary Material). (B) Same comparison as (A) in individuals with active infection. (C) Production of IFNγ and IL-17 from murine OT-II cells cocultured with OVA peptide and splenic CD11c+CD11b+ FACS sorted from the spleens of naïve mice or mice infected for 5 weeks with BCG in the presence of a polyclonal antibody specific to DLL4 or IgG control antibody. Cells were cultured for 72 h in the presence of 10 μg/mL anti-DLL4 or control antibody. One-way ANOVA analysis indicated DLL4 was a significant factor in determining cytokine output from T cells. For IFNγ, the p value was <0.0001, and for IL-17 the p value was 0.02.
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Figure 4: Delta-like 4 expression correlates with cytokine production from T cells during latent infection. (A) Linear regression analysis of CD4+ T cell IFNγ production, as measured by intracellular cytokine staining, compared to the percent of CD14+ monocytes expressing DLL4 in individuals diagnosed with latent Mtb infection. Similar results were obtained with IL-2 and TNFα production (Figure S4 in Supplementary Material). (B) Same comparison as (A) in individuals with active infection. (C) Production of IFNγ and IL-17 from murine OT-II cells cocultured with OVA peptide and splenic CD11c+CD11b+ FACS sorted from the spleens of naïve mice or mice infected for 5 weeks with BCG in the presence of a polyclonal antibody specific to DLL4 or IgG control antibody. Cells were cultured for 72 h in the presence of 10 μg/mL anti-DLL4 or control antibody. One-way ANOVA analysis indicated DLL4 was a significant factor in determining cytokine output from T cells. For IFNγ, the p value was <0.0001, and for IL-17 the p value was 0.02.
Mentions: Notch ligand expression on APCs has been shown to alter the host immune response to several pathogens (35–39). For example, we have previously demonstrated that T cell-derived IL-17 production is increased as a result of Dll4 expression in a model of mycobacterial infection (31). To determine if DLL4 expression altered cytokine production from T cells in humans infected with M. tuberculosis, we assessed the CD4 T cell production of TNFα, IFNγ, and IL-2 in response to characterized mycobacterial antigens including ESAT-6, TB10.4, Ag85A, CFP-10, and PPD by intracellular cytokine staining in both actively and latently infected individuals. We also assessed the production of these same cytokines in response to the superantigen Staphylococcal Enterotoxin B (SEB) as a positive control. We then performed PCA using these data, including the percentage of DLL4+ monocytes as a factor to determine if DLL4 expression in monocytes was correlated with cytokine production from T cells in latently and actively infected individuals. PCA analysis is routinely used to reduce the number of dimensions in a data set by determining if specific variables track together as a component (40). Our analysis of latently infected individuals resulted in two components each with an Eigenvalue ≥4. The values in Table 1 state that the correlation between each variable and the two discovered principal components on a scale of 1 to −1, with 1 being a 100% correlation. Values of less than 0.3 are considered non-significant. Based on this analysis, we determined that DLL4 expression was correlated with cytokine production from CD4 T cells as a result of stimulation with specific TB antigens including ESAT-6 and PPD in an antigen-specific component. The second component contained all of the cytokine data related to SEB stimulation and was not correlated with DLL4 expression on monocytes. Interestingly, CFP-10 cytokine secretion was associated with the SEB component, suggesting a different pattern of cytokine secretion than that induced by the other antigens. Based on our component matrix (Table S1 in Supplementary Material), which correlates all variables in the data set, we determined that DLL4 expression was most closely correlated with the cytokine response following stimulation with PPD (Table S1 in Supplementary Material). Table S2 in Supplementary Material states that the component containing SEB cytokine stimulation was not related to the component containing DLL4 expression and cytokine production in response to TB antigens. Performing dimension reduction analysis on individuals diagnosed with active TB disease did not result in a component containing DLL4 expression. This analysis informed us that DLL4 expression on monocytes was associated with T cell cytokine production in latently infected individuals. We then performed linear regression analysis to determine if there was a direct correlation between cytokine production and DLL4 expression in both latent infection and active TB disease. Our results indicate an r2 value of 0.54 when correlating the percent of CD4+ T cells producing IFNγ and the percentage of monocytes expressing DLL4 in response to PPD. There was no correlation between DLL4 expression and cytokine production in response to SEB (Figure 4A). There was no correlation between cytokine production and DLL4 expression on monocytes in patients with active TB disease (Figure 4B). There was also no correlation between CD8+ T cell cytokine production and DLL4 expression (Table S3 in Supplementary Material). Consistent with previous data, there was also reduced frequency of cytokine-producing T cells in patients with active TB (33).

View Article: PubMed Central - PubMed

ABSTRACT

The Notch ligand delta-like 4 (DLL4) is known to fine-tune the CD4+ T cell cytokine response. DLL4 is expressed on the surface of antigen-presenting cells (APCs) in a MyD88-dependent manner. We found that DLL4 expression was upregulated on bone marrow progenitor cells and APCs in mice infected with BCG Mycobacterium. Transfer of DLL4+ progenitor cells from infected hosts resulted in an increase DLL4+ myeloid cells in the spleen, indicating that expression of the dll4 gene is propagated throughout hematopoiesis. We also found an increase in DLL4+ monocytes from individuals who were infected with Mycobacterium tuberculosis. In latent individuals, DLL4 expression correlated with increased cytokine production from T cells in response to PPD stimulation. Finally, antibody blockade of DLL4 reduced T cell cytokine production from na&iuml;ve T cells stimulated with antigen. These results demonstrate that the Notch ligand DLL4 can influence T cell cytokine production in both humans and mice, and further reveal that expression of DLL4 is upregulated on early hematopoietic progenitors in response to chronic mycobacterial infection. These data suggest that widespread DLL4 expression may occur as a result of mycobacterial infection, and that this expression may alter CD4+ T cell responses to both previously encountered and novel antigens.

No MeSH data available.


Related in: MedlinePlus