Myeloid-derived suppressor cells contribute to the subversion of innate immunity during Staphylococcus aureus biofilm infection

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature monocytes and granulocytes that are potent inhibitors of T cell activation. A role for MDSCs in bacterial infections has only recently emerged, and our laboratory was the first to demonstrate a functional role for MDSCs during Staphylococcus aureus (S. aureus) biofilm infection. Biofilm infections often lead to significant morbidity due to their recalcitrance to antibiotics and ability to subvert immune-mediated clearance by skewing the immune response toward an anti-inflammatory, pro-fibrotic phenotype. Therefore, we examined whether MDSCs could play a role in this process. CD11b+Gr-1+ MDSCs represented the main cellular infiltrate during S. aureus orthopedic biofilm infection, and biofilm-associated MDSCs inhibited T cells proliferation and cytokine production, which correlated with a paucity of T cell infiltrates at the infection site. Importantly, tissues obtained from patients undergoing revision surgery for prosthetic joint infections (PJIs) revealed similar patterns of immune cell influx, with increased MDSC-like infiltrates and significantly fewer T cells compared to aseptic revisions. Depletion of MDSCs and improved bacterial clearance by enhancing the intrinsic proinflammatory attributes of infiltrating monocytes and macrophages. However, the mechanisms responsible for MDSC homing to sites of biofilm infection and factors mediating immunosuppression remain unknown. In cancer, proinflammatory signals initially induce MDSC recruitment and activation, while the immunosuppressive functions of MDSCs are mediated through factors like IL-10, Arg-1 and iNOS. IL-12p40 and IL-10 are both significantly elevated during S. aureus biofilm infection. These studies demonstrate that IL-12 plays a key role in the recruitment of MDSCs into biofilm infection via a chemoattractant that remains to be identified, while IL-10 is produced by infiltrating MDSCs at the site of biofilm infection, whereupon it plays a critical role in polarizing monocyte/macrophages toward an anti-inflammatory phenotype. Loss of either IL-12 or IL-10 during the early MDSC recruitment or effector phases, respectively, promotes biofilm clearance, implicating key roles for each cytokine at distinct stages of infection. Collectively, these studies demonstrate that MDSCs are key contributors to the chronicity of S. aureus biofilm infection, as their immunosuppressive function prevents monocyte/macrophage proinflammatory activity, which facilitates biofilm persistence

Molecular pathways elicited by Toll-like receptor 2 (TLR2) signaling during Staphylococcus aureus craniotomy infection

A craniotomy is required to access the brain during neurosurgery for tumor resection or epilepsy treatment and despite extensive precautionary measures, infectious complications occur at a frequency of 1-3% (1-3). Approximately half of craniotomy infections are caused by S. aureus, which forms a biofilm on the bone flap. Our laboratory has developed a mouse model of S. aureus craniotomy-associated biofilm infection that shares important ultrastructural and MRI attributes with human disease (4). Toll-like receptor 2 (TLR2) is expressed by cells of the innate immune system and is critical for recognizing pathogen-associated molecular patterns (PAMPs) in the cell wall of gram-positive bacteria, such as S. aureus. Recent studies have shown that TLR2 is critical for S. aureus containment during craniotomy infection, in that TLR2 knockout (KO) mice displayed increased bacterial burden in the brain, galea, and bone flap during acute and chronic infection (days 3 and 14, respectively)(5). Cytokine and chemokine expression was dramatically reduced in TLR2 KO mice which did not coincide with a decrease in leukocyte infiltrates in the brain or galea. This suggested that S. aureus outgrowth in the context of TLR2 deficiency is not the result of altered leukocyte recruitment, but instead due to defects in their activation status. To determine this, RNA-sequencing (RNA-seq) was performed on microglia purified from the brain of WT and TLR2 KO mice at either day 3 or 7 post-infection by FACS with the goal of identifying microglial transcripts that are divergent between the two mouse strains (either increased or decreased). RNA-seq revealed Fibroblast Growth Factor Receptor (FGFR) 2 and 3 were upregulated in TLR2 KO microglia. This suggests a direct correlation between TLR2, FGFR2 and FGFR3. Since TLR2 KO animals have a bias toward an anti-inflammatory response to infection and the fact that FGFR2 and FGFR3 are upregulated during wound healing or infection, this could be a reason why higher levels are observed in TLR2 KO microglia. FGRF2 and FGRF3 are key regulators of neuronal protection and repair following brain injury/infection. Therefore, microglia may be increasing the expression of these receptors in an effort to control the damage that is occurring in response to the increased bacterial burden in TLR2 KO animals.https://digitalcommons.unmc.edu/surp2020/1000/thumbnail.jp

Hiding in Plain Sight: Interplay between Staphylococcal Biofilms and Host Immunity.

Staphylococcus aureus and Staphylococcus epidermidis are notable for their propensity to form biofilms on implanted medical devices. Staphylococcal biofilm infections are typified by their recalcitrance to antibiotics and ability to circumvent host immune-mediated clearance, resulting in the establishment of chronic infections that are often recurrent in nature. Indeed, the immunomodulatory lifestyle of biofilms seemingly shapes the host immune response to ensure biofilm engraftment and persistence in an immune competent host. Here, we provide a brief review of the mechanisms whereby S. aureus and S. epidermidis biofilms manipulate host-pathogen interactions and discuss the concept of microenvironment maintenance in infectious outcomes, as well as speculate how these findings pertain to the challenges of staphylococcal vaccine development

TLR2 and Caspase-1 Signaling are Critical for Bacterial Containment But Not Clearance During Craniotomy-Associated Biofilm Infection

BACKGROUND: A craniotomy is required to access the brain for tumor resection or epilepsy treatment, and despite precautionary measures, infectious complications occur at a frequency of 1-3%. Approximately half of craniotomy infections are caused by Staphylococcus aureus (S. aureus) that forms a biofilm on the bone flap, which is recalcitrant to antibiotics. Our prior work in a mouse model of S. aureus craniotomy infection revealed a critical role for myeloid differentiation factor 88 (MyD88) in bacterial containment and pro-inflammatory mediator production. Since numerous receptors utilize MyD88 as a signaling adaptor, the current study examined the importance of Toll-like receptor 2 (TLR2) and TLR9 based on their ability sense S. aureus ligands, namely lipoproteins and CpG DNA motifs, respectively. We also examined the role of caspase-1 based on its known association with TLR signaling to promote IL-1β release. METHODS: A mouse model of craniotomy-associated biofilm infection was used to investigate the role of TLR2, TLR9, and caspase-1 in disease progression. Wild type (WT), TLR2 knockout (KO), TLR9 KO, and caspase-1 KO mice were examined at various intervals post-infection to quantify bacterial burden, leukocyte recruitment, and inflammatory mediator production in the galea, brain, and bone flap. In addition, the role of TLR2-dependent signaling during microglial/macrophage crosstalk with myeloid-derived suppressor cells (MDSCs) was examined. RESULTS: TLR2, but not TLR9, was important for preventing S. aureus outgrowth during craniotomy infection, as revealed by the elevated bacterial burden in the brain, galea, and bone flap of TLR2 KO mice concomitant with global reductions in pro-inflammatory mediator production compared to WT animals. Co-culture of MDSCs with microglia or macrophages, to model interactions in the brain vs. galea, respectively, also revealed a critical role for TLR2 in triggering pro-inflammatory mediator production. Similar to TLR2, caspase-1 KO animals also displayed increased S. aureus titers coincident with reduced pro-inflammatory mediator release, suggestive of pathway cooperativity. Treatment of caspase-1 KO mice with IL-1β microparticles significantly reduced S. aureus burden in the brain and galea compared to empty microparticles, confirming the critical role of IL-1β in limiting S. aureus outgrowth during craniotomy infection. CONCLUSIONS: These results demonstrate the existence of an initial anti-bacterial response that depends on both TLR2 and caspase-1 in controlling S. aureus growth; however, neither pathway is effective at clearing infection in the WT setting, since craniotomy infection persists when both molecules are present

Monocyte Metabolic Reprogramming Promotes Pro-Inflammatory Activity and Staphylococcus Aureus Biofilm Clearance

Biofilm-associated prosthetic joint infections (PJIs) cause significant morbidity due to their recalcitrance to immune-mediated clearance and antibiotics, with Staphylococcus aureus (S. aureus) among the most prevalent pathogens. We previously demonstrated that S. aureus biofilm-associated monocytes are polarized to an anti-inflammatory phenotype and the adoptive transfer of pro-inflammatory macrophages attenuated biofilm burden, highlighting the critical role of monocyte/macrophage inflammatory status in dictating biofilm persistence. The inflammatory properties of leukocytes are linked to their metabolic state, and here we demonstrate that biofilm-associated monocytes exhibit a metabolic bias favoring oxidative phosphorylation (OxPhos) and less aerobic glycolysis to facilitate their anti-inflammatory activity and biofilm persistence. To shift monocyte metabolism in vivo and reprogram cells to a pro-inflammatory state, a nanoparticle approach was utilized to deliver the OxPhos inhibitor oligomycin to monocytes. Using a mouse model of S. aureus PJI, oligomycin nanoparticles were preferentially internalized by monocytes, which significantly reduced S. aureus biofilm burden by altering metabolism and promoting the pro-inflammatory properties of infiltrating monocytes as revealed by metabolomics and RT-qPCR, respectively. Injection of oligomycin alone had no effect on monocyte metabolism or biofilm burden, establishing that intracellular delivery of oligomycin is required to reprogram monocyte metabolic activity and that oligomycin lacks antibacterial activity against S. aureus biofilms. Remarkably, monocyte metabolic reprogramming with oligomycin nanoparticles was effective at clearing established biofilms in combination with systemic antibiotics. These findings suggest that metabolic reprogramming of biofilm-associated monocytes may represent a novel therapeutic approach for PJI

Staphylococcus aureus ATP Synthase Promotes Biofilm Persistence by Influencing Innate Immunity

ABSTRACT Staphylococcus aureus is a major cause of prosthetic joint infection (PJI), which is characterized by biofilm formation. S. aureus biofilm skews the host immune response toward an anti-inflammatory profile by the increased recruitment of myeloid-derived suppressor cells (MDSCs) that attenuate macrophage proinflammatory activity, leading to chronic infection. A screen of the Nebraska Transposon Mutant Library identified several hits in the ATP synthase operon that elicited a heightened inflammatory response in macrophages and MDSCs, including atpA, which encodes the alpha subunit of ATP synthase. An atpA transposon mutant (ΔatpA) had altered growth kinetics under both planktonic and biofilm conditions, along with a diffuse biofilm architecture that was permissive for leukocyte infiltration, as observed by confocal laser scanning microscopy. Coculture of MDSCs and macrophages with ΔatpA biofilm elicited significant increases in the proinflammatory cytokines interleukin 12p70 (IL-12p70), tumor necrosis factor alpha (TNF-α), and IL-6. This was attributed to increased leukocyte survival resulting from less toxin and protease production by ΔatpA biofilm as determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The enhanced inflammatory response elicited by ΔatpA biofilm was cell lysis-dependent since it was negated by polyanethole sodium sulfanate treatment or deletion of the major autolysin, Atl. In a mouse model of PJI, ΔatpA-infected mice had decreased MDSCs concomitant with increased monocyte/macrophage infiltrates and proinflammatory cytokine production, which resulted in biofilm clearance. These studies identify S. aureus ATP synthase as an important factor in influencing the immune response during biofilm-associated infection and bacterial persistence. IMPORTANCE Medical device-associated biofilm infections are a therapeutic challenge based on their antibiotic tolerance and ability to evade immune-mediated clearance. The virulence determinants responsible for bacterial biofilm to induce a maladaptive immune response remain largely unknown. This study identified a critical role for S. aureus ATP synthase in influencing the host immune response to biofilm infection. An S. aureus ATP synthase alpha subunit mutant (ΔatpA) elicited heightened proinflammatory cytokine production by leukocytes in vitro and in vivo, which coincided with improved biofilm clearance in a mouse model of prosthetic joint infection. The ability of S. aureus ΔatpA to augment host proinflammatory responses was cell lysis-dependent, as inhibition of bacterial lysis by polyanethole sodium sulfanate or a ΔatpAΔatl biofilm did not elicit heightened cytokine production. These studies reveal a critical role for AtpA in shaping the host immune response to S. aureus biofilm

Neutrophils are Mediators of Metastatic Prostate Cancer Progression in Bone

Bone metastatic prostate cancer (BM-PCa) significantly reduces overall patient survival and is currently incurable. Current standard immunotherapy showed promising results for PCa patients with metastatic, but less advanced, disease (i.e., fewer than 20 bone lesions) suggesting that PCa growth in bone contributes to response to immunotherapy. We found that: (1) PCa stimulates recruitment of neutrophils, the most abundant immune cell in bone, and (2) that neutrophils heavily infiltrate regions of prostate tumor in bone of BM-PCa patients. Based on these findings, we examined the impact of direct neutrophil-prostate cancer interactions on prostate cancer growth. Bone marrow neutrophils directly induced apoptosis of PCa in vitro and in vivo, such that neutrophil depletion in bone metastasis models enhanced BM-PCa growth. Neutrophil-mediated PCa killing was found to be mediated by suppression of STAT5, a transcription factor shown to promote PCa progression. However, as the tumor progressed in bone over time, neutrophils from late-stage bone tumors failed to elicit cytotoxic effector responses to PCa. These findings are the first to demonstrate that bone-resident neutrophils inhibit PCa and that BM-PCa are able to progress via evasion of neutrophil-mediated killing. Enhancing neutrophil cytotoxicity in bone may present a novel therapeutic option for bone metastatic prostate cancer