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Mucosal T-helper 17 Responses to Chlamydia Genital Tract Infection
S138 Abstracts
524
SUNDAY
A Functional IL-13 Receptor is Expressed on Polarized Murine CD4 1 Th17 Cells and IL-13 Signaling Negatively Regulates Th17 Cytokine Production D. C. Newcomb1, W. Zhou1, M. L. Moore1, K. Goleniewska1, G. K. K. Hershey2, J. K. Kolls3, R. S. Peebles, Jr1; 1Vanderbilt. University Medical Center, Nashville, TN, 2Cincinnati Children’s Hospital, Cincinnati, OH, 3 University of Pittsburgh, Pittsburgh, PA. RATIONALE: Th17 cells are involved in autoimmune and inflammatory disorders, produce IL-17A and IL-21, and are negatively regulated by the Th2 cytokine, IL-4, and the Th1 cytokine, IFN-g. We show for the first time that CD4 1 Th17 cells express mRNA for the IL-13 receptor (IL13R). Based on the inhibitory role IL-4 has on IL-17A production in polarized CD4 1 Th17 cells, we hypothesized that IL-13, another Th2 cytokine, would attenuate IL-17A production. METHODS: CD41 splenocytes from BALB/c, IL-4 KO, or IL-4R KO mice were activated with anti-CD3 and anti-CD28 and polarized into Th17 cells using TGF-b, IL-6, and IL-23 in the presence of IL-13 (010ng/ml). Four days after Th17 polarization cultured supernatants, total RNA, or total protein were collected and examined. RESULTS: Th17 cells increased expression of IL-13Ra1 and IL-4Ra, components of IL-13R, compared to naı¨ ve T cells. The addition of IL-13 at the time of Th17 polarization attenuated IL-17A and IL-21 expression. Th17 cells from IL-4 KO mice, but not IL-4R KO mice, had IL-13induced inhibition of IL-17A expression. The addition of IL-13 during polarization on WT Th17 cells increased IL-13R expression, downregulated ROR-gT expression, the transcription required for Th17 development, upregulated expression of GATA3, the transcription factor activated during the development of Th2 cells, and induced STAT6 phosphorylation. CONCLUSIONS: Th17 cells express a functional IL-13R allowing for an IL-13-induced increase in receptor expression and STAT6 phosphorylation. IL-13 also negatively regulates IL-17A and IL-21 production. Therefore, using therapeutics to inhibit IL-13 production could have adverse consequences by upregulating Th17 inflammation in certain disease states.
525
Effect of Retinoic Acid on the Expression of the Receptors that Regulate B-cell Homeostasis: TACI, BAFF-R, and BCMA A. A. Lieberman, M. Ballow; SUNY at Buffalo School of Medicine and Biomedical Sciences, Buffalo, NY. RATIONALE: all-trans retinoic acid (atRA) is known to augment immunoglobulin (Ig) secretion by B cells, however, the mechanism(s) by which atRA modulates this immune response remains unknown. TACI, BAFF-R and BMCA are receptors of the TNF superfamily that are expressed by B cells at varying levels throughout cell maturation and are involved in regulating B-cell homeostasis. We examined the effects of atRA on the expression of these B cell receptors to determine if the known effect of atRA on Ig production is associated with changes in B-cell-surface expression profiles. METHODS: PBMC from healthy adults were isolated, and B cells were purified by negative selection using immunomagnetic beads. PBMC or isolated B-cells were cultured for 72 hours with varying concentrations of atRA (10-8 M-10-6 M), and in the presence or absence of Staphylococcus Aureus Cowan strain I (SAC), a polyclonal B-cell activator. TACI, BAFF-R and BMCA expression on B-cells were analyzed by flow cytometry. RESULTS: B cell surface expression of TACI and BCMA increased in a dose-dependant manner with the addition of atRA when activated by SAC (n 5 7 experiments, 10-7 M and 10-6 M (p < 0.01)). BAFF-R was constitutively expressed on 95% of B-cells, and did not vary with atRA treatment. The increased expression of TACI and BCMA by atRA was observed in both isolated B cells as well as B cells in mixed PBMC culture. CONCLUSIONS: atRA modulates the expression of TACI and BCMA on B cells in both mixed and isolated B cell cultures in the presence of SAC activation.
J ALLERGY CLIN IMMUNOL FEBRUARY 2009
526
Mucosal T-helper 17 Responses to Chlamydia Genital Tract Infection A. M. Scurlock1, C. M. O’Connell2, C. W. Andrews3, I. P. Foote1, T. M. Darville2; 1University of Arkansas for Medical Sciences/Arkansas Children’s Hospital Research Institute, Little Rock, AR, 2University of Pittsburgh Medical Center/Children’s Hospital of Pittsburgh, Pittsburgh, PA, 3 Rockdale Medical Center, Conyers, GA. RATIONALE: Although Interferon-gamma (IFNg)-producing CD4 1 Thelper (Th)-1 cells are critical to resolve Chlamydia genital tract (GT) infection, these cells, along with neutrophils, have been implicated in development of GT pathology. We examined the neutrophil-inducing CD4 1 Th17 response as an alternative mechanism of chlamydial immunopathogenesis. METHODS: Mucosal immune responses and histopathologic outcomes were compared in female IFNg knockout (IFNgKO) and C57/Bl6 control mice following intra-vaginal Chlamydia muridarum infection. GT secretions and iliac node (ILN) cell supernatants were assayed for Th17 associated cytokines/chemokines by ELISA or cytokine bead array. Following CD4 enrichment, IL-17 and IFNg producing GT cells were quantified by ELISPOT assay. RESULTS: When compared to controls, IFNgKO mice demonstrated enhanced GT pathology and neutrophilic infiltrate (p < 0.05), but equivalent lymphocytic infiltrates despite an absent Th1/IFNg response. Both groups displayed C. muridarum-specific lymphocyte proliferation that was attenuated by anti-CD4. IL-17 was detected in ILN supernatants from both groups but was significantly higher in IFNgKO (p < 0.001) and was abrogated in both groups by anti-CD4. IFNgKO mice exhibited higher IL-17 levels in genital tract secretions from days 7-35 post-infection (p < 0.001), while controls had higher IL-17 levels from days 2-5 (p < 0.05). Significant increases in IL-6 (p < 0.001), TGFb (p < 0.05), TNFa (p < 0.001), and the neutrophil chemokine CXCL1/KC (p < 0.001) were detected in IFNgKO mice. Both IFNg and IL-17 are induced in C57/Bl6 controls, with CD4 1 IL-17 producing cells detected by ELISPOT on days 4, 10, 20 post-infection. CONCLUSIONS: These data support counter-regulation between genital mucosal Th1 and Th17 responses and suggest a novel mechanism for Chlamydia-associated GT pathology through Th17-mediated neutrophil influx.
527
Image-Based Flow Cytometry as a Tool for Analyzing CD251CD41T Cells in Atopic Subjects A. J. Reefer, M. D. Solga, J. A. Lannigan, J. A. Woodfolk; University of Virginia, Charlottesville, VA. RATIONALE: Studies on the role of circulating CD251CD41 T cells in allergic disease in humans have been hampered by the similar molecular signature of regulatory T cells (Tregs) and activated effectors. New approaches to dissecting this complex cell population are warranted. Here, we report how flow cytometry imaging may provide new insight into the role of CD251 T cells at sites of allergic inflammation. METHODS: CD41 T cells isolated from highly atopic subjects (total IgE >1,000 IU/ml) were stimulated with ‘‘pro-allergic’’ factors (thymic stromal lymphopoietin (TSLP) 1 allergen (Der p 1)) using autologous dendritic cells as APCs. T cells were analyzed (day 7) by image-based flow cytometry (Amnis ImagestreamÒ) after staining for surface (CD25, CD127, TSLP receptor) and intracellular (Foxp3 and IL-4) markers. Dividing T cells were identified by counterstaining with DNA binding dyes. RESULTS: CD251 T cells induced by ‘‘pro-allergic’’ factors comprised a mixture consistent with Tregs (CD127lo) and effectors (CD127hi). Both populations of CD251 T cells were actively dividing and single-cell imaging revealed a larger cellular area as compared with CD25neg T cells, consistent with blasting T cells. Surprisingly, co-expression of IL-4 and Foxp3 was a feature of dividing CD127lo T cells, as well as CD127hi T cells. Moreover, imaging revealed that surface expression of TSLP receptor co-segregated with high expression of IL-4 in a subset of CD127lo T cells. CONCLUSIONS: Flow cytometry imaging of dividing T cells suggests that in a pro-allergic milieu, Th2 effectors could masquerade as CD127lo Tregs and these cells are armed to respond directly to TSLP.
524
SUNDAY
A Functional IL-13 Receptor is Expressed on Polarized Murine CD4 1 Th17 Cells and IL-13 Signaling Negatively Regulates Th17 Cytokine Production D. C. Newcomb1, W. Zhou1, M. L. Moore1, K. Goleniewska1, G. K. K. Hershey2, J. K. Kolls3, R. S. Peebles, Jr1; 1Vanderbilt. University Medical Center, Nashville, TN, 2Cincinnati Children’s Hospital, Cincinnati, OH, 3 University of Pittsburgh, Pittsburgh, PA. RATIONALE: Th17 cells are involved in autoimmune and inflammatory disorders, produce IL-17A and IL-21, and are negatively regulated by the Th2 cytokine, IL-4, and the Th1 cytokine, IFN-g. We show for the first time that CD4 1 Th17 cells express mRNA for the IL-13 receptor (IL13R). Based on the inhibitory role IL-4 has on IL-17A production in polarized CD4 1 Th17 cells, we hypothesized that IL-13, another Th2 cytokine, would attenuate IL-17A production. METHODS: CD41 splenocytes from BALB/c, IL-4 KO, or IL-4R KO mice were activated with anti-CD3 and anti-CD28 and polarized into Th17 cells using TGF-b, IL-6, and IL-23 in the presence of IL-13 (010ng/ml). Four days after Th17 polarization cultured supernatants, total RNA, or total protein were collected and examined. RESULTS: Th17 cells increased expression of IL-13Ra1 and IL-4Ra, components of IL-13R, compared to naı¨ ve T cells. The addition of IL-13 at the time of Th17 polarization attenuated IL-17A and IL-21 expression. Th17 cells from IL-4 KO mice, but not IL-4R KO mice, had IL-13induced inhibition of IL-17A expression. The addition of IL-13 during polarization on WT Th17 cells increased IL-13R expression, downregulated ROR-gT expression, the transcription required for Th17 development, upregulated expression of GATA3, the transcription factor activated during the development of Th2 cells, and induced STAT6 phosphorylation. CONCLUSIONS: Th17 cells express a functional IL-13R allowing for an IL-13-induced increase in receptor expression and STAT6 phosphorylation. IL-13 also negatively regulates IL-17A and IL-21 production. Therefore, using therapeutics to inhibit IL-13 production could have adverse consequences by upregulating Th17 inflammation in certain disease states.
525
Effect of Retinoic Acid on the Expression of the Receptors that Regulate B-cell Homeostasis: TACI, BAFF-R, and BCMA A. A. Lieberman, M. Ballow; SUNY at Buffalo School of Medicine and Biomedical Sciences, Buffalo, NY. RATIONALE: all-trans retinoic acid (atRA) is known to augment immunoglobulin (Ig) secretion by B cells, however, the mechanism(s) by which atRA modulates this immune response remains unknown. TACI, BAFF-R and BMCA are receptors of the TNF superfamily that are expressed by B cells at varying levels throughout cell maturation and are involved in regulating B-cell homeostasis. We examined the effects of atRA on the expression of these B cell receptors to determine if the known effect of atRA on Ig production is associated with changes in B-cell-surface expression profiles. METHODS: PBMC from healthy adults were isolated, and B cells were purified by negative selection using immunomagnetic beads. PBMC or isolated B-cells were cultured for 72 hours with varying concentrations of atRA (10-8 M-10-6 M), and in the presence or absence of Staphylococcus Aureus Cowan strain I (SAC), a polyclonal B-cell activator. TACI, BAFF-R and BMCA expression on B-cells were analyzed by flow cytometry. RESULTS: B cell surface expression of TACI and BCMA increased in a dose-dependant manner with the addition of atRA when activated by SAC (n 5 7 experiments, 10-7 M and 10-6 M (p < 0.01)). BAFF-R was constitutively expressed on 95% of B-cells, and did not vary with atRA treatment. The increased expression of TACI and BCMA by atRA was observed in both isolated B cells as well as B cells in mixed PBMC culture. CONCLUSIONS: atRA modulates the expression of TACI and BCMA on B cells in both mixed and isolated B cell cultures in the presence of SAC activation.
J ALLERGY CLIN IMMUNOL FEBRUARY 2009
526
Mucosal T-helper 17 Responses to Chlamydia Genital Tract Infection A. M. Scurlock1, C. M. O’Connell2, C. W. Andrews3, I. P. Foote1, T. M. Darville2; 1University of Arkansas for Medical Sciences/Arkansas Children’s Hospital Research Institute, Little Rock, AR, 2University of Pittsburgh Medical Center/Children’s Hospital of Pittsburgh, Pittsburgh, PA, 3 Rockdale Medical Center, Conyers, GA. RATIONALE: Although Interferon-gamma (IFNg)-producing CD4 1 Thelper (Th)-1 cells are critical to resolve Chlamydia genital tract (GT) infection, these cells, along with neutrophils, have been implicated in development of GT pathology. We examined the neutrophil-inducing CD4 1 Th17 response as an alternative mechanism of chlamydial immunopathogenesis. METHODS: Mucosal immune responses and histopathologic outcomes were compared in female IFNg knockout (IFNgKO) and C57/Bl6 control mice following intra-vaginal Chlamydia muridarum infection. GT secretions and iliac node (ILN) cell supernatants were assayed for Th17 associated cytokines/chemokines by ELISA or cytokine bead array. Following CD4 enrichment, IL-17 and IFNg producing GT cells were quantified by ELISPOT assay. RESULTS: When compared to controls, IFNgKO mice demonstrated enhanced GT pathology and neutrophilic infiltrate (p < 0.05), but equivalent lymphocytic infiltrates despite an absent Th1/IFNg response. Both groups displayed C. muridarum-specific lymphocyte proliferation that was attenuated by anti-CD4. IL-17 was detected in ILN supernatants from both groups but was significantly higher in IFNgKO (p < 0.001) and was abrogated in both groups by anti-CD4. IFNgKO mice exhibited higher IL-17 levels in genital tract secretions from days 7-35 post-infection (p < 0.001), while controls had higher IL-17 levels from days 2-5 (p < 0.05). Significant increases in IL-6 (p < 0.001), TGFb (p < 0.05), TNFa (p < 0.001), and the neutrophil chemokine CXCL1/KC (p < 0.001) were detected in IFNgKO mice. Both IFNg and IL-17 are induced in C57/Bl6 controls, with CD4 1 IL-17 producing cells detected by ELISPOT on days 4, 10, 20 post-infection. CONCLUSIONS: These data support counter-regulation between genital mucosal Th1 and Th17 responses and suggest a novel mechanism for Chlamydia-associated GT pathology through Th17-mediated neutrophil influx.
527
Image-Based Flow Cytometry as a Tool for Analyzing CD251CD41T Cells in Atopic Subjects A. J. Reefer, M. D. Solga, J. A. Lannigan, J. A. Woodfolk; University of Virginia, Charlottesville, VA. RATIONALE: Studies on the role of circulating CD251CD41 T cells in allergic disease in humans have been hampered by the similar molecular signature of regulatory T cells (Tregs) and activated effectors. New approaches to dissecting this complex cell population are warranted. Here, we report how flow cytometry imaging may provide new insight into the role of CD251 T cells at sites of allergic inflammation. METHODS: CD41 T cells isolated from highly atopic subjects (total IgE >1,000 IU/ml) were stimulated with ‘‘pro-allergic’’ factors (thymic stromal lymphopoietin (TSLP) 1 allergen (Der p 1)) using autologous dendritic cells as APCs. T cells were analyzed (day 7) by image-based flow cytometry (Amnis ImagestreamÒ) after staining for surface (CD25, CD127, TSLP receptor) and intracellular (Foxp3 and IL-4) markers. Dividing T cells were identified by counterstaining with DNA binding dyes. RESULTS: CD251 T cells induced by ‘‘pro-allergic’’ factors comprised a mixture consistent with Tregs (CD127lo) and effectors (CD127hi). Both populations of CD251 T cells were actively dividing and single-cell imaging revealed a larger cellular area as compared with CD25neg T cells, consistent with blasting T cells. Surprisingly, co-expression of IL-4 and Foxp3 was a feature of dividing CD127lo T cells, as well as CD127hi T cells. Moreover, imaging revealed that surface expression of TSLP receptor co-segregated with high expression of IL-4 in a subset of CD127lo T cells. CONCLUSIONS: Flow cytometry imaging of dividing T cells suggests that in a pro-allergic milieu, Th2 effectors could masquerade as CD127lo Tregs and these cells are armed to respond directly to TSLP.