Granulocytic myeloid-derived suppressor cell activity during biofilm infection is regulated by a glycolysis/HIF1a axis.

Staphylococcus aureus is a leading cause of biofilm-associated prosthetic joint infection (PJI). A primary contributor to infection chronicity is an expansion of granulocytic myeloid-derived suppressor cells (G-MDSCs), which are critical for orchestrating the antiinflammatory biofilm milieu. Single-cell sequencing and bioinformatic metabolic algorithms were used to explore the link between G-MDSC metabolism and S. aureus PJI outcome. Glycolysis and the hypoxia response through HIF1a were significantly enriched in G-MDSCs. Interfering with both pathways in vivo, using a 2-deoxyglucose nanopreparation and granulocyte-targeted Hif1a conditional KO mice, respectively, attenuated G-MDSC-mediated immunosuppression and reduced bacterial burden in a mouse model of S. aureus PJI. In addition, single-cell RNA-Seq (scRNA-Seq) analysis of granulocytes from PJI patients also showed an enrichment in glycolysis and hypoxia-response genes. These findings support the importance of a glycolysis/HIF1a axis in promoting G-MDSC antiinflammatory activity and biofilm persistence during PJI.

Elucidating granulocytic myeloid-derived suppressor cell heterogeneity during Staphylococcus aureus biofilm infection.

Myeloid-derived suppressor cells (MDSCs) are pathologically activated immature myeloid cells with immunosuppressive activity that expand during chronic inflammation, such as cancer and prosthetic joint infection (PJI). MDSCs can be broadly separated into two populations based on surface marker expression and function, namely monocytic MDSCs (M-MDSCs) and granulocytic MDSCs (G-MDSCs). G-MDSCs are the most abundant leukocyte infiltrate during PJI; however, how this population is maintained in vivo and cellular heterogeneity is currently unknown. In this study, we identified a previously unknown population of Ly6G + Ly6C + F4/80 + MHCII+ MDSCs during PJI that displayed immunosuppressive properties ex vivo. We leveraged F4/80 and MHCII expression by these cells for further characterization using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), which revealed a distinct transcriptomic signature of this population. F4/80 + MHCII+ MDSCs displayed gene signatures resembling G-MDSCs, neutrophils, and monocytes, but had significantly increased expression of pathways involved in cytokine response/production, inflammatory cell death, and mononuclear cell differentiation. To determine whether F4/80 + MHCII+ MDSCs represented an alternate phenotypic state of G-MDSCs, Ly6G + Ly6C + F4/80-MHCII- G-MDSCs from CD45.1 mice were adoptively transferred into CD45.2 recipients using a mouse model of PJI. A small percentage of transferred G-MDSCs acquired F4/80 and MHCII expression in vivo, suggesting some degree of plasticity in this population. Collectively, these results demonstrate a previously unappreciated phenotype of F4/80 + MHCII+ MDSCs during PJI, revealing that a granulocytic-to-monocytic transition can occur during biofilm infection.

A critical role for staphylococcal nitric oxide synthase in controlling flavohemoglobin toxicity.

Most coagulase-negative staphylococcal species, including the opportunistic pathogen Staphylococcus epidermidis, struggle to maintain redox homeostasis and grow under nitrosative stress. Under these conditions, growth can only resume once nitric oxide (NO) is detoxified by the flavohemoglobin Hmp. Paradoxically, S. epidermidis produces endogenous NO through its genetically encoded nitric oxide synthase (seNOS) and heavily relies on its activity for growth. In this study, we investigate the basis of the growth advantage attributed to seNOS activity. Our findings reveal that seNOS supports growth by countering Hmp toxicity. S. epidermidis relies on Hmp activity for its survival in the host under NO stress. However, in the absence of nitrosative stress, Hmp generates significant amounts of the harmful superoxide radical (O2•-) from its heme prosthetic group which impedes growth. To limit Hmp toxicity, nitrite (NO2-) derived from seNOS promotes CymR-CysK regulatory complex activity, which typically regulates cysteine metabolism, but we now demonstrate to also repress hmp transcription. These findings reveal a critical mechanism through which the bacterial NOS-Hmp axis drives staphylococcal fitness.

IL-10 production by granulocytes promotes Staphylococcus aureus craniotomy infection.

BACKGROUND: Treatment of brain tumors, epilepsy, or hemodynamic abnormalities requires a craniotomy to access the brain. Nearly 1 million craniotomies are performed in the US annually, which increase to ~ 14 million worldwide and despite prophylaxis, infectious complications after craniotomy range from 1 to 3%. Approximately half are caused by Staphylococcus aureus (S. aureus), which forms a biofilm on the bone flap that is recalcitrant to antibiotics and immune-mediated clearance. However, the mechanisms responsible for the persistence of craniotomy infection remain largely unknown. The current study examined the role of IL-10 in promoting bacterial survival. METHODS: A mouse model of S. aureus craniotomy infection was used with wild type (WT), IL-10 knockout (KO), and IL-10 conditional KO mice where IL-10 was absent in microglia and monocytes/macrophages (CX3CR1CreIL-10 fl/fl) or neutrophils and granulocytic myeloid-derived suppressor cells (G-MDSCs; Mrp8CreIL-10 fl/fl), the major immune cell populations in the infected brain vs. subcutaneous galea, respectively. Mice were examined at various intervals post-infection to quantify bacterial burden, leukocyte recruitment, and inflammatory mediator production in the brain and galea to assess the role of IL-10 in craniotomy persistence. In addition, the role of G-MDSC-derived IL-10 on neutrophil activity was examined. RESULTS: Granulocytes (neutrophils and G-MDSCs) were the major producers of IL-10 during craniotomy infection. Bacterial burden was significantly reduced in IL-10 KO mice in the brain and galea at day 14 post-infection compared to WT animals, concomitant with increased CD4+ and γδ T cell recruitment and cytokine/chemokine production, indicative of a heightened proinflammatory response. S. aureus burden was reduced in Mrp8CreIL-10 fl/fl but not CX3CR1CreIL-10 fl/fl mice that was reversed following treatment with exogenous IL-10, suggesting that granulocyte-derived IL-10 was important for promoting S. aureus craniotomy infection. This was likely due, in part, to IL-10 production by G-MDSCs that inhibited neutrophil bactericidal activity and TNF production. CONCLUSION: Collectively, these findings reveal a novel role for granulocyte-derived IL-10 in suppressing S. aureus clearance during craniotomy infection, which is one mechanism to account for biofilm persistence.

Transcriptional Profiling of Phagocytic Leukocytes and Microglia Reveals a Critical Role for Reactive Oxygen Species in Biofilm Containment during Staphylococcus aureus Craniotomy Infection.

Craniotomies are performed to treat a variety of intracranial pathology. Surgical site infection remains a complication of craniotomy despite the use of prophylactic antibiotics and universal sterile precautions. Infections occur in 1-3% of procedures, with approximately half caused by Staphylococcus aureus that forms a biofilm on the bone flap and is recalcitrant to systemic antibiotic therapy. We used an S. aureus-dsRed construct to compare the phagocytic capacity of leukocytes and microglia in vitro and in vivo using a mouse model of craniotomy infection. In addition, single-cell RNA sequencing (scRNA-seq) was applied to determine whether a transcriptional signature could be identified for phagocytic versus nonphagocytic cells in vivo. S. aureus was phagocytosed to equivalent extents in microglia, macrophages, neutrophils, and granulocytic myeloid-derived suppressor cells in vitro; however, microglial uptake of S. aureus was limited in vivo, whereas the other leukocyte populations exhibited phagocytic activity. scRNA-seq comparing the transcriptional signatures of phagocytic (S. aureus-dsRed+) versus nonphagocytic (S. aureus-dsRed-) leukocytes identified classical pathways enriched in phagocytic cells (i.e., reactive oxygen species [ROS]/reactive nitrogen species, lysosome, iron uptake, and transport), whereas nonphagocytic populations had increased ribosomal, IFN, and hypoxia signatures. scRNA-seq also revealed a robust ROS profile, which led to the exploration of craniotomy infection in NADPH oxidase 2 knockout mice. S. aureus burden, leukocyte recruitment, and intracellular bacterial load were significantly increased in NADPH oxidase 2 KO compared with wild-type animals. Collectively, these results highlight the importance of ROS generation in phagocytes for S. aureus biofilm containment, but not clearance, during craniotomy infection.

Role of Staphylococcus aureus Formate Metabolism during Prosthetic Joint Infection.

Biofilms are bacterial communities characterized by antibiotic tolerance. Staphylococcus aureus is a leading cause of biofilm infections on medical devices, including prosthetic joints, which represent a significant health care burden. The major leukocyte infiltrate associated with S. aureus prosthetic joint infection (PJI) is granulocytic myeloid-derived suppressor cells (G-MDSCs), which produce IL-10 to promote biofilm persistence by inhibiting monocyte and macrophage proinflammatory activity. To determine how S. aureus biofilm responds to G-MDSCs and macrophages, biofilms were cocultured with either leukocyte population followed by RNA sequencing. Several genes involved in fermentative pathways were significantly upregulated in S. aureus biofilm following G-MDSC coculture, including formate acetyltransferase (pflB), which catalyzes the conversion of pyruvate and coenzyme-A into formate and acetyl-CoA. A S. aureus pflB mutant (ΔpflB) did not exhibit growth defects in vitro. However, ΔpflB formed taller and more diffuse biofilm compared to the wild-type strain as revealed by confocal microscopy. In a mouse model of PJI, the bacterial burden was significantly reduced with ΔpflB during later stages of infection, which coincided with decreased G-MDSC influx and increased neutrophil recruitment, and ΔpflB was more susceptible to macrophage killing. Although formate was significantly reduced in the soft tissue surrounding the joint of ΔpflB-infected mice levels were increased in the femur, suggesting that host-derived formate may also influence bacterial survival. This was supported by the finding that a ΔpflBΔfdh strain defective in formate production and catabolism displayed a similar phenotype to ΔpflB. These results revealed that S. aureus formate metabolism is important for promoting biofilm persistence.

Transcriptional Diversity and Niche-Specific Distribution of Leukocyte Populations during Staphylococcus aureus Craniotomy-Associated Biofilm Infection.

Neurosurgery for brain tumor resection or epilepsy treatment requires a craniotomy to gain access to the brain. Despite prophylactic measures, infectious complications occur at a frequency of 1-3%, with approximately half caused by Staphylococcus aureus (S. aureus) that forms a biofilm on the bone flap and is recalcitrant to antibiotics. Using single-cell RNA sequencing in a mouse model of S. aureus craniotomy infection, this study revealed the complex transcriptional heterogeneity of resident microglia and infiltrating monocytes in the brain, in addition to transcriptionally diverse granulocyte subsets in the s.c. galea and bone flap. In the brain, trajectory analysis identified the transition of microglia from a homeostatic/anti-inflammatory to proinflammatory and proliferative populations, whereas granulocytes in the brain demonstrated a trajectory from a granulocyte myeloid-derived suppressor cell (MDSC)-like phenotype to a small population of mature polymorphonuclear neutrophils (PMNs). In the galea, trajectory analysis identified the progression from two distinct granulocyte-MDSC-like populations to PMN clusters enriched for IFN signaling and cell cycle genes. Based on their abundance in the galea and bone flap, PMNs and MDSCs were depleted using anti-Ly6G, which resulted in increased bacterial burden. This revealed a critical role for PMNs in S. aureus containment because MDSCs were found to attenuate PMN antibacterial activity, which may explain, in part, why craniotomy infection persists in the presence of PMN infiltrates. These results demonstrate the existence of a transcriptionally diverse leukocyte response that likely influences the chronicity of S. aureus craniotomy infection.

Synthesis and SAR Studies of 1H-Pyrrolo[2,3-b]pyridine-2-carboxamides as Phosphodiesterase 4B (PDE4B) Inhibitors.

Herein we report the synthesis, SAR, and biological evaluation of a series of 1H-pyrrolo[2,3-b]pyridine-2-carboxamide derivatives as selective and potent PDE4B inhibitors. Compound 11h is a PDE4B preferring inhibitor and exhibited acceptable in vitro ADME and significantly inhibited TNF-α release from macrophages exposed to pro-inflammatory stimuli (i.e., lipopolysaccharide and the synthetic bacterial lipopeptide Pam3Cys). In addition, 11h was selective against a panel of CNS receptors and represents an excellent lead for further optimization and preclinical testing in the setting of CNS diseases.

Staphylococcus aureus ATP Synthase Promotes Biofilm Persistence by Influencing Innate Immunity.

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.

Orthopaedic Surgery Elicits a Systemic Anti-Inflammatory Signature.

Little information is available on the functional activity of leukocytes after arthroplasty or the expansion of populations with immune suppressive properties during the acute post-operative period. Synovial fluid and matched pre- and post-surgical blood samples were collected from total hip and knee arthroplasty patients (THA and TKA, respectively) to examine the impact of surgery on peripheral blood leukocyte frequency, bactericidal activity, and inflammatory mediator expression. For spinal surgeries, inflammatory mediator production by peripheral blood mononuclear cells (PBMCs) pre- and post-surgery was examined. An expansion of immune suppressive granulocytic myeloid-derived suppressor cells (G-MDSCs) was observed following arthroplasty, which correlated with significantly increased serum interleukin-10 (IL-10) levels. Analysis of synovial fluid from THA and TKAs revealed reduced granulocyte colony-stimulating factor (G-CSF) and soluble CD40 ligand (sCD40L) and increased interleukin-6 (IL-6), monocyte chemoattractant protein 2 (CCL2) and Fms-like tyrosine kinase 3 ligand (Flt-3L) compared to pre- and post-surgical serum. For the spinal surgery cohort, stimulation of PBMCs isolated post-surgery with bacterial antigens produced significantly less pro-inflammatory (IL-1α, IL-1ß, interleukin-1 receptor antagonist (IL-1RA), IL-12p40, growth-related oncogene-α/GRO-α (CXCL1) and 6Ckine (CCL21)) and more anti-inflammatory/tissue repair mediators (IL-10, G-CSF and granulocyte-macrophage colony-stimulating factor (GM-CSF)) compared to PBMCs recovered before surgery. The observed bias towards systemic anti-inflammatory changes without concomitant increases in pro-inflammatory responses may influence susceptibility to infection following orthopaedic surgery in the context of underlying co-morbidities or risk factors.

Lactate production by Staphylococcus aureus biofilm inhibits HDAC11 to reprogramme the host immune response during persistent infection.

Staphylococcus aureus is a leading cause of biofilm-associated prosthetic joint infection (PJI), resulting in considerable disability and prolonged treatment. It is known that host leukocyte IL-10 production is required for S. aureus biofilm persistence in PJI. An S. aureus bursa aurealis Tn library consisting of 1,952 non-essential genes was screened for mutants that failed to induce IL-10 in myeloid-derived suppressor cells (MDSCs), which identified a critical role for bacterial lactic acid biosynthesis. We generated an S. aureus ddh/ldh1/ldh2 triple Tn mutant that cannot produce D- or L-lactate. Co-culture of MDSCs or macrophages with ddh/ldh1/ldh2 mutant biofilm produced substantially less IL-10 compared with wild-type S. aureus, which was also observed in a mouse model of PJI and led to reduced biofilm burden. Using MDSCs recovered from the mouse PJI model and in vitro leukocyte-biofilm co-cultures, we show that bacterial-derived lactate inhibits histone deacetylase 11, causing unchecked HDAC6 activity and increased histone 3 acetylation at the Il-10 promoter, resulting in enhanced Il-10 transcription in MDSCs and macrophages. Finally, we show that synovial fluid of patients with PJI contains elevated amounts of D-lactate and IL-10 compared with control subjects, and bacterial lactate increases IL-10 production by human monocyte-derived macrophages.

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-1ß 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-1ß microparticles significantly reduced S. aureus burden in the brain and galea compared to empty microparticles, confirming the critical role of IL-1ß 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.

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.

Heterogeneity of Ly6G+ Ly6C+ Myeloid-Derived Suppressor Cell Infiltrates during Staphylococcus aureus Biofilm Infection.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature monocytes and granulocytes. While neutrophils (polymorphonuclear leukocytes [PMNs]) are classically identified as highly differentiated cells specialized for antimicrobial defense, our laboratory has reported minor contributions of PMNs to the immune response during Staphylococcusaureus biofilm infection. However, these two cell types can be difficult to differentiate because of shared surface marker expression. Here we describe a more refined approach to distinguish MDSCs from PMNs utilizing the integrin receptor CD11b combined with conventional Ly6G and Ly6C expression. This approach separated the Ly6G+ Ly6C+ population that we previously identified in a mouse model of S. aureus orthopedic implant infection into two subsets, namely, CD11bhigh Ly6G+ Ly6C+ MDSCs and CD11blow Ly6G+ Ly6C+ PMNs, which was confirmed by characteristic nuclear morphology using cytospins. CD11bhigh Ly6G+ Ly6C+ MDSCs suppressed T cell proliferation throughout the 28-day infection period, whereas CD11blow Ly6G+ Ly6C+ PMNs had no effect early (day 3 postinfection), although this population acquired suppressive activity at later stages of biofilm development. To further highlight the distinctions between biofilm-associated MDSCs and PMNs versus monocytes, transcriptional profiles were compared by transcriptome sequencing (RNA-Seq). A total of 6,466 genes were significantly differentially expressed in MDSCs versus monocytes, whereas only 297 genes were significantly different between MDSCs and PMNs. A number of genes implicated in cell cycle regulation were identified, and in vivo ethynyldeoxyuridine (EdU) labeling revealed that approximately 50% of MDSCs proliferated locally at the site of S. aureus biofilm infection. Based on their similar transcriptomic profiles to those of PMNs, biofilm-associated MDSCs are of a granulocytic lineage and can be classified as granulocytic MDSCs (G-MDSCs).

Arginase-1 Expression in Myeloid Cells Regulates Staphylococcus aureus Planktonic but Not Biofilm Infection.

Staphylococcus aureus is a leading cause of device-associated biofilm infections, which represent a serious health care concern based on their chronicity and antibiotic resistance. We previously reported that S. aureus biofilms preferentially recruit myeloid-derived suppressor cells (MDSCs), which promote monocyte and macrophage anti-inflammatory properties. This is associated with increased myeloid arginase-1 (Arg-1) expression, which has been linked to anti-inflammatory and profibrotic activities that are observed during S. aureus biofilm infections. To determine whether MDSCs and macrophages utilize Arg-1 to promote biofilm infection, Arg-1 was deleted in myeloid cells by use of Tie-2Cre mice. Despite Arg-1 expression in biofilm-associated myeloid cells, bacterial burdens and leukocyte infiltrates were similar between wild-type (WT) and Arg-1fl/fl;Tie-2Cre conditional knockout (KO) mice from days 3 to 14 postinfection in both orthopedic implant and catheter-associated biofilm models. However, inducible nitric oxide synthase (iNOS) expression was dramatically elevated in biofilm-associated MDSCs from Arg-1fl/fl;Tie-2Cre animals, suggesting a potential Arg-1-independent compensatory mechanism for MDSC-mediated immunomodulation. Treatment of Arg-1fl/fl;Tie-2Cre mice with the iNOS inhibitor N6-(1-iminoethyl)-l-lysine (l-NIL) had no effect on biofilm burdens or immune infiltrates, whereas treatment of WT mice with the Arg-1/ornithine decarboxylase inhibitor difluoromethylornithine (DFMO) increased bacterial titers, but only in the surrounding soft tissues, which possess attributes of a planktonic environment. A role for myeloid-derived Arg-1 in regulating planktonic infection was confirmed using a subcutaneous abscess model, in which S. aureus burdens were significantly increased in Arg-1fl/fl;Tie-2Cre mice compared to those in WT mice. Collectively, these results indicate that the effects of myeloid Arg-1 are context dependent and are manifest during planktonic but not biofilm infection.

Human prosthetic joint infections are associated with myeloid-derived suppressor cells (MDSCs): Implications for infection persistence.

Prosthetic joint infection (PJI) is a devastating complication of joint arthroplasty surgery typified by biofilm formation. Currently, mechanisms whereby biofilms persist and evade immune-mediated clearance in immune competent patients remain largely ill-defined. Therefore, the current study characterized leukocyte infiltrates and inflammatory mediator expression in tissues from patients with PJI compared to aseptic loosening. CD33+ HLA-DR- CD66b+ CD14-/low granulocytic myeloid-derived suppressor cells (G-MDSCs) were the predominant leukocyte population at sites of human PJI compared to aseptic tissues. MDSCs inhibit T cell proliferation, which coincided with reduced T cells in PJIs compared to aseptic tissues. IL-10, IL-6, and CXCL1 were significantly elevated in PJI tissues and have been implicated in MDSC inhibitory activity, expansion, and recruitment, respectively, which may account for their preferential increase in PJIs. This bias towards G-MDSC accumulation during human PJI could account for the chronicity of these infections by preventing the pro-inflammatory, antimicrobial actions of immune effector cells. CLINICAL SIGNIFICANCE: Animal models of PJI have revealed a critical role for MDSCs and IL-10 in promoting infection persistence; however, whether this population is prevalent during human PJI and across distinct bacterial pathogens remains unknown. This study has identified that granulocytic-MDSC infiltrates are unique to human PJIs caused by distinct bacteria, which are not associated with aseptic loosening of prosthetic joints. Better defining the immune status of human PJIs could lead to novel immune-mediated approaches to facilitate PJI clearance in combination with conventional antibiotics. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1605-1613, 2018.

Indoleacrylic Acid Produced by Commensal Peptostreptococcus Species Suppresses Inflammation.

Host factors in the intestine help select for bacteria that promote health. Certain commensals can utilize mucins as an energy source, thus promoting their colonization. However, health conditions such as inflammatory bowel disease (IBD) are associated with a reduced mucus layer, potentially leading to dysbiosis associated with this disease. We characterize the capability of commensal species to cleave and transport mucin-associated monosaccharides and identify several Clostridiales members that utilize intestinal mucins. One such mucin utilizer, Peptostreptococcus russellii, reduces susceptibility to epithelial injury in mice. Several Peptostreptococcus species contain a gene cluster enabling production of the tryptophan metabolite indoleacrylic acid (IA), which promotes intestinal epithelial barrier function and mitigates inflammatory responses. Furthermore, metagenomic analysis of human stool samples reveals that the genetic capability of microbes to utilize mucins and metabolize tryptophan is diminished in IBD patients. Our data suggest that stimulating IA production could promote anti-inflammatory responses and have therapeutic benefits.

Interleukin-10 production by myeloid-derived suppressor cells contributes to bacterial persistence during Staphylococcus aureus orthopedic biofilm infection.

Staphylococcus aureus is known to establish biofilms on medical devices. We recently demonstrated that Ly6G(high)Ly6C(+) myeloid-derived suppressor cells are critical for allowing S. aureus biofilms to subvert immune-mediated clearance; however, the mechanisms whereby myeloid-derived suppressor cells promote biofilm persistence remain unknown. Interleukin-10 expression was significantly increased in a mouse model of S. aureus orthopedic implant biofilm infection with kinetics that mirrored myeloid-derived suppressor cell recruitment. Because myeloid-derived suppressor cells produce interleukin-10, we explored whether it was involved in orchestrating the nonproductive immune response that facilitates biofilm formation. Analysis of interleukin-10-green fluorescent protein reporter mice revealed that Ly6G(high)Ly6C(+) myeloid-derived suppressor cells were the main source of interleukin-10 during the first 2 wk of biofilm infection, whereas monocytes had negligible interleukin-10 expression until day 14. Myeloid-derived suppressor cell influx into implant-associated tissues was significantly reduced in interleukin-10 knockout mice at day 14 postinfection, concomitant with increased monocyte and macrophage infiltrates that displayed enhanced proinflammatory gene expression. Reduced myeloid-derived suppressor cell recruitment facilitated bacterial clearance, as revealed by significant decreases in S. aureus burdens in the knee joint, surrounding soft tissue, and femur of interleukin-10 knockout mice. Adoptive transfer of interleukin-10 wild-type myeloid-derived suppressor cells into S. aureus-infected interleukin-10 knockout mice restored the local biofilm-permissive environment, as evidenced by increased bacterial burdens and inhibition of monocyte proinflammatory activity. These effects were both interleukin-10-dependent and interleukin-10-independent because myeloid-derived suppressor cell-derived interleukin-10 was required for promoting biofilm growth and anti-inflammatory gene expression in monocytes but was not involved in monocyte recruitment to biofilm-infected tissues. These results demonstrate that interleukin-10 production by myeloid-derived suppressor cells contributes to the persistence of S. aureus orthopedic biofilm infections.

IL-12 promotes myeloid-derived suppressor cell recruitment and bacterial persistence during Staphylococcus aureus orthopedic implant infection.

Staphylococcus aureus is a leading cause of human prosthetic joint infections (PJIs) typified by biofilm formation. We recently identified a critical role for myeloid-derived suppressor cells (MDSCs) in S. aureus biofilm persistence. Proinflammatory signals induce MDSC recruitment and activation in tumor models; however, the mechanisms responsible for MDSC homing to sites of biofilm infection are unknown. In this study, we report that several cytokines (IL-12p40, IL-1ß, TNF-α, and G-CSF) and chemokines (CXCL2, CCL5) were significantly elevated in a mouse model of S. aureus PJI. This coincided with significantly increased MDSC infiltrates concomitant with reduced monocyte, macrophage, and T cell influx compared with uninfected animals. Of the cytokines detected, IL-12 was of particular interest based on its ability to possess either pro- or anti-inflammatory effects mediated through p35-p40 heterodimers or p40 homodimers, respectively. MDSC recruitment was significantly reduced in both p40 and p35 knockout mice, which resulted in enhanced monocyte and neutrophil influx and bacterial clearance. Adoptive transfer of wild-type MDSCs into infected p40 knockout animals worsened disease outcome, as evidenced by the return of S. aureus burdens to levels typical of wild-type mice. Tissues obtained from patients undergoing revision surgery for PJI revealed similar patterns of immune cell influx, with increased MDSC-like cells and significantly fewer T cells compared with aseptic revisions. These findings reveal a critical role for IL-12 in shaping the anti-inflammatory biofilm milieu by promoting MDSC recruitment.