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Volume 22, Issue 4, Pages e4 (October 2017)

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1 Volume 22, Issue 4, Pages 543-551.e4 (October 2017)
O-Acetylation of Peptidoglycan Limits Helper T Cell Priming and Permits Staphylococcus aureus Reinfection  Marisel Sanchez, Stacey L. Kolar, Sabrina Müller, Christopher N. Reyes, Andrea J. Wolf, Chihiro Ogawa, Rajat Singhania, Daniel D. De Carvalho, Moshe Arditi, David M. Underhill, Gislâine A. Martins, George Y. Liu  Cell Host & Microbe  Volume 22, Issue 4, Pages e4 (October 2017) DOI: /j.chom Copyright © 2017 Elsevier Inc. Terms and Conditions

2 Cell Host & Microbe 2017 22, 543-551. e4DOI: (10. 1016/j. chom. 2017
Copyright © 2017 Elsevier Inc. Terms and Conditions

3 Figure 1 Prior S. aureus Infection Does Not Induce Protective Immunity against Reinfection (A) C57BL/6 mice (n = 8–11) were injected i.p. with 107 CFUs of S. aureus (LAC USA300) GAS (M49), or PBS on day 0, and re-injected on day 21 with 107 CFUs of either S. aureus or GAS. Bacteria from spleen and kidneys were recovered after 24 hr. PBS/S. aureus (Sa) injected with PBS on day 0 and then injected with SA on day 21. Dashed lines indicate limit of detection. (B) C57BL/6 mice (n = 10) were injected i.p. with S. aureus (107), GAS (5 × 106), or PBS. The mice were inoculated after 21 days with S. aureus (4 × 107) or GAS (2 × 107) and monitored for survival. (C) C57BL/6 mice (n = 5) were infected i.p. once, twice (day 0 and 21), or four times (day 0, 21, 28, and 35) with S. aureus (107). Surviving CFUs were enumerated 24 hr after the last infection. (D) Mice (n = 5–6) were infected as in (A). Splenocytes were harvested 7 days after the last infection, stimulated with phorbol 12-myristate 13-acetate, ionomycin, and brefeldin A for 4 hr, and stained for surface CD4 and T cell receptor β (TCR-β), and intracellular IFN-γ and IL-17A. Shown are fluorescence-activated cell sorting plots and graphs of IFN-γ and IL-17A expression by CD4+TCR-β+ cells. Results are representative of two experiments. (E) Mice (n = 8–11) were infected as in (A), and splenocytes harvested 7 days after the last infection were stimulated with heat-killed S. aureus or GAS (MOI = 10) for 48 hr. Supernatants were analyzed by ELISA. Data are representative of two experiments. See also Figure S1. For (A), (C), and (D), each data point represents an individual mouse, bars denote median and dashed lines indicate the limit of detection. In (E), data are shown as mean (bars) and SEM (error bars). Data analysis was performed using Mann-Whitney U test for (A and D), ANOVA for (C), log rank test for (B) and Student's t test for (E). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant. Cell Host & Microbe  , e4DOI: ( /j.chom ) Copyright © 2017 Elsevier Inc. Terms and Conditions

4 Figure 2 S. aureus Induces Suboptimal Polarization of TH1 and TH17 Cells Associated with Differential Regulation of Gene Expression in DCs (A and B) BMDCs were cultured with medium only (NS, non-stimulated), S. aureus (LAC), or GAS (MOI = 3) for 1 hr, pulsed with OVA peptide (6 hr) and then mixed with carboxyfluorescein succinimidyl ester (CFSE)-labeled OT-II transgenic CD4+ T cells. (A) CFSE dilution in gated CD4+ T cells at day 7 post stimulation and (B) IFN-γ and IL-17A in the culture supernatants after 7 days. (C) MHC II, CD80, and CD86 expression on BMDCs after 24 hr of stimulation with GAS or S. aureus (MOI = 10). (D) BMDCs were left without any stimulation (NS) or incubated with S. aureus or GAS (MOI = 3) for 8 hr and total RNA was extracted for RNA sequencing analysis. Top graph: Venn diagrams showing genes up- (left) or downregulated (right) by S. aureus only (pink), GAS only (green), or both GAS and S. aureus (brown). Selected genes are listed for each group. Bottom graph: fold change (over NS) for selected genes shown in (D). (E) Cytokine production as measured by ELISA in the supernatant of DCs treated as in (D) for 24 hr. All data are representative of at least three experiments except for the IL-23 ELISA, which was performed twice. In (A–C and E), data are shown as means (bars) and SEM (error bars) and data analysis was performed using ANOVA. ∗p < 0.05; ∗∗p < See also Figure S2 and Tables S1 and S2. Cell Host & Microbe  , e4DOI: ( /j.chom ) Copyright © 2017 Elsevier Inc. Terms and Conditions

5 Figure 3 O-Acetylation of S. aureus PGN Limits the Induction of Pro-inflammatory Cytokines Critical for Protective Memory (A) BMDCs were stimulated with WT (S. aureus SA113), ΔoatA (isogenic SA113 mutant), or a 1:1 mixture of WT and ΔoatA at an MOI of 10. Supernatants were harvested at 24 hr for cytokine determination. Data are representative of three experiments. See also Figure S3. (B and C) Mice (n = 5) were infected with WT S. aureus (2 × 107) or WT + ΔoatA (107 CFUs of each) at day 0, 7, and 14. (B) Two days after the last infection, splenocytes were harvested from the mice for determination of cytokine levels. (C) Seven days after the last infection, splenocytes were harvested and stimulated with heat-killed WT S. aureus and cytokines were measured after 3 days. For (B) and (C), each data point represents an individual mouse, and means ± SEM are shown for all data. In (A), data are shown as means (bars) and SEM (erros bars). Data analysis was performed using Student's t test (A) and using Mann-Whitney U test for (B and C). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < Cell Host & Microbe  , e4DOI: ( /j.chom ) Copyright © 2017 Elsevier Inc. Terms and Conditions

6 Figure 4 Adjuvancy with ΔoatA or Absence of IL-10 Promotes Development of Protective Immunity to S. aureus Reinfection (A) Mice (n = 6–7) were injected with PBS or infected three times as in (Figure 3B). Ten days after the last injection, all mice were inoculated with WT SA113 S. aureus (2 × 107). CFUs from the kidneys were enumerated after 24 hr. The data are representative of two experiments. (B) WT or IL-17A−/− mice were infected three times with S. aureus as in (Figure 3B). Seven days after the last infection, splenocytes were harvested and 5 × 107 purified total CD4+ T cells were transferred intravenously (i.v.) into naive mice (n = 5–6). The recipient mice were infected with WT S. aureus (2 × 107) the next day and kidney CFUs were enumerated after 24 hr. See also Figure S4. (C–E) WT or IL-10−/− mice (n = 4–6) were infected with WT S. aureus (SA113) three times at 7 day intervals. A non-pretreated group served as a control. Seven days after the last pretreatment, all groups of mice were inoculated with WT S. aureus (2 × 107). (C) Spleen cytokine levels 24 hr after the last infection. (D) Splenocytes harvested 24 hr after the last infection were stimulated with heat-killed S. aureus (MOI = 3), and cytokines in the supernatants were assessed after 3 days. (E) CFUs from splenocytes harvested 24 hr after the last infection. (F) WT or IL-10−/− mice were infected three times with S. aureus SA113 as in (Figure 3B). Seven days after the last infection, splenocytes were harvested and 5 × 107 purified total CD4+ T cells were transferred i.v. into naive mice (n = 5–6). The recipient mice were infected with WT S. aureus (2 × 107) the next day and kidney CFUs were enumerated after 24 hr. For (A), (B), (E), and (F), each data point represents an individual mouse. In (C), data are shown as means (bars) and SEM (error bars). Data analysis was performed using ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < Cell Host & Microbe  , e4DOI: ( /j.chom ) Copyright © 2017 Elsevier Inc. Terms and Conditions


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