Control of adaptive immune responses by Staphylococcus aureus through IL-10, PD-L1, and TLR2

Abstract

Microbes induce innate immune responses in hosts. It is critical to know how different microbes control adaptive responses through innate pathways. The impact of gram-positive bacteria on the innate and adaptive responses is unclear. Herein we report that Staphylococcus aureus induces IL-10, Th17-inducing cytokines IL-6 and IL-23, chemokines, and regulates dendritic cell markers. S. aureus inhibits T-cell IL-2 responses through modulation of HLA-DR, CD86 and PD-L1. IFN-gamma, Src kinase inhibitors, or TLR2 antibodies prevented the down-modulation of HLA-DR by S. aureus. Our data demonstrate that innate TLR signaling induces multi-dimensional inhibition of adaptive immune responses, which may contribute to the lack of protective immunity to bacteria or microbe tolerance. IL-10 and PD-L1 antagonists may boost immunity to vaccines for S. aureus and other microbes.

Figure 1. Effects of S. aureus on the expression of HLA-DR and CD86 in human monocytes.

(a) and (b). Monocytes were cultured in macrophage-SFM (Med) or in the presence of SAC (0.0075% or 0.0075g/100mL). After 40 hours, cells were analyzed for the expression of indicated cell surface molecules. Each marker was assessed for 3 to 10 times with similar results. The number at corner of each histogram is the value of GMFI (geometric mean fluorescence intensity). (c) and (d) Monocytes were cultured in the presence of different doses of SAC and stained for HLA-DR and CD86. Cells were subject to acquisition and analysis on flow cytometer. (e) Statistic analysis of the down modulation of HLA-DR and CD86 (data from 8 donors) by S. aureus. (f). Monocytes were cultured in medium and stimulated with LTA, or LPS for 40 hours.

Figure 2. Induction of cytokines and chemokines in human monocytes by S. aureus.

Secretion of cytokines. Monocytes were pre-stimulated with or without IFN-γ for 16 hours, then stimulated with SAC (0.0075%) overnight. Cytokines were measured by specific ELISAs with supernatants from 5 to 8 donors. Results represented as mean (pink line) of each group in box plots. (a) TNF-α and IL-12. (b) IL-10. (c) Transcriptional Induction of cytokines and chemokines by SAC. Monocytes (5 x 10⁶ /well) were stimulated with SAC for 5 hours. Total RNA was isolated and RT-PCR was carried out as described in Materials and Methods. Data were from one donor representing three donors with similar results. (d) Transcriptional induction of cytokines by SAC for indicated times in the absence or presence of IFN-γ.

Figure 3. Role of TLR2 in the downmodulation of MHC Class II molecules.

Human primary monocytes were treated for 40 hours with S. aureus with/without one hour pre-incubation with indicated blocking antibodies. First row: isotype control. Cells from donor A were also used for experiments shown in Figure 1f.

Figure 4. HLA-DR down-modulation by S. aureus: role of IL-10 and kinases.

Monocytes were cultured in medium in the presence of: (a) indicated reagents (IL-10, 20 ng/mL; IFN-γ, 100 ng/ml and SAC) for 40 hours. Data is representive of results with 3 donors. (b) in the presence of SAC with or without anti-IL-10 neutralizing antibodies or anti-TNF-α neutralizing antibodies for 40 hours. Cells were stained, washed and analyzed on flow cytometer for the expression of CD86, HLA-DR, and CD36. Two additional experiments have been done with similar results. (c) Monocytes were cultured in the presence or absence of SAC for 60 hours. After fixation and permeablization cells were stained with indicated antibodies on coverslips and subject to analysis with the confocal microscope equipped with an objective lens of 63x. Overlay: FITC, DAPI, DIC. (d) Monocytes were cultured in medium and pretreated with genistein (G, 74 μM), herbimycin A (H, 1 μg/ml), or tyrphostin (T, 1 μg/ml) for one hour, then stimulated with SAC for 40 hours. Data were pooled data from three donors. (e) Monocytes were pretreated with indicated inhibitors for five hours, then treated with SAC for 40 hours. Cells were stained with indicated antibodies and subject to analysis on flow cytometer. HerbA, herbimycin A; Src KI, Src kinase inhibitor II; GMFI, geometric mean fluorescence intensity. (f) Induction of NF-κB nuclear translocation in human monocytes by S. aureus. (A) Monocytes (25 x 10⁶/well) were stimulated with either LPS, or SAC for 1 hour. Nuclear extracts (NE) were made and assayed according to Materials and Methods. Data were from one donor representing two with similar results. (B) Nuclear extracts were made as (A) and anti-p50 antibody or anti-p65 antibody was added to the DNA binding reactions and assayed according to Materials and Methods.

Figure 5. Induction of PD-L1 by SAC.

Monocytes were treated with SAC (0.75%) and stained with indicated markers. (a) Expression of HLA-DR and co-stimulatory or co-inhibitory molecules in the presence or absence of indicated reagents. The data presented are from one donor, are representive of results from three different donors. (b) Dose dependent induction of PD-L1 by SAC.

Figure 6. Control of T cell IL-2 response by SAC and restoration by anti-IL-10 and anti-PD-L1.

PBLs and allo-macrophages from multiple pairs of donors were cultured as described in Materials and Methods. (a) T cell IL-2 responses at 24 hours of mixed leukocyte reaction in the presence or absence of SAC as determined by specific IL-2 ELISA. (b) and (c) T cell IL-2 responses at 24 hours of mixed leukocyte reaction in the presence or absence of SAC and indicated neutralizing anti-IL-10 antibodies, or anti-PD-L1, anti-PD-L2 blocking antibodies. (d) Proposed model of microbe tolerance.