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Weak organic acids are commonly found in host niches colonized by bacteria, and they can inhibit bacterial growth as the environment becomes acidic. This inhibition is often attributed to the toxicity resulting from the accumulation of high concentrations of organic anions in the cytosol, which disrupts cellular homeostasis. However, the precise cellular targets that organic anions poison and the mechanisms used to counter organic anion intoxication in bacteria have not been elucidated. Here, we utilize acetic acid, a weak organic acid abundantly found in the gut to investigate its impact on the growth of Staphylococcus aureus . We demonstrate that acetate anions bind to and inhibit D-alanyl-D-alanine ligase (Ddl) activity in S. aureus . Ddl inhibition reduces intracellular D-alanyl-D-alanine (D-Ala-D-Ala) levels, compromising staphylococcal peptidoglycan cross-linking and cell wall integrity. To overcome the effects of acetate-mediated Ddl inhibition, S. aureus maintains a substantial intracellular D-Ala pool through alanine racemase (Alr1) activity and additionally limits the flux of D-Ala to D-glutamate by controlling D-alanine aminotransferase (Dat) activity. Surprisingly, the modus operandi of acetate intoxication in S. aureus is common to multiple biologically relevant weak organic acids indicating that Ddl is a conserved target of small organic anions. These findings suggest that S. aureus may have evolved to maintain high intracellular D-Ala concentrations, partly to counter organic anion intoxication., Significance: Under mildly acidic conditions, weak organic acids like acetic acid accumulate to high concentrations within the cytosol as organic anions. However, the physiological consequence of organic anion accumulation is poorly defined. Here we investigate how the acetate anion impacts S. aureus . We show that acetate anions directly bind Ddl and inhibit its activity. The resulting decrease in intracellular D-Ala-D-Ala pools impacts peptidoglycan integrity. Since acetate is a weak inhibitor of Ddl, mechanisms that maintain a high intracellular D-Ala pools are sufficient to counter the effect of acetate-mediated Ddl inhibition in S. aureus .

Background: Environmental lipopolysaccharide (LPS) and microbial component-enriched organic dusts cause significant lung disease. These environmental exposures induce the recruitment and activation of distinct lung monocyte/macrophage subpopulations involved in disease pathogenesis. Aconitate decarboxylase 1 ( Acod1 ) was one of the most upregulated genes following LPS (vs. saline) exposure of murine whole lungs with transcriptomic profiling of sorted lung monocyte/macrophage subpopulations also highlighting its significance. Given monocyte/macrophage activation can be tightly linked to metabolism, the objective of these studies was to determine the role of the immunometabolic regulator ACOD1 in environmental exposure-induced lung inflammation., Methods: Wild-type (WT) mice were intratracheally (i.t.) instilled with 10 μg of LPS or saline. Whole lungs were profiled using bulk RNA sequencing or sorted to isolate monocyte/macrophage subpopulations. Sorted subpopulations were then characterized transcriptomically using a NanoString innate immunity multiplex array 48 h post-exposure. Next, WT and Acod1 -/- mice were instilled with LPS, 25% organic dust extract (ODE), or saline, whereupon serum, bronchoalveolar lavage fluid (BALF), and lung tissues were collected. BALF metabolites of the tricarboxylic acid (TCA) cycle were quantified by mass spectrometry. Cytokines/chemokines and tissue remodeling mediators were quantitated by ELISA. Lung immune cells were characterized by flow cytometry. Invasive lung function testing was performed 3 h post-LPS with WT and Acod1 -/- mice., Results: Acod1 -/- mice treated with LPS demonstrated decreased BALF levels of itaconate, TCA cycle reprogramming, decreased BALF neutrophils, increased lung CD4 + T cells, decreased BALF and lung levels of TNF-α, and decreased BALF CXCL1 compared to WT animals. In comparison, Acod1 -/- mice treated with ODE demonstrated decreased serum pentraxin-2, BALF levels of itaconate, lung total cell, neutrophil, monocyte, and B-cell infiltrates with decreased BALF levels of TNF-α and IL-6 and decreased lung CXCL1 vs. WT animals. Mediators of tissue remodeling (TIMP1, MMP-8, MMP-9) were also decreased in the LPS-exposed Acod1 -/- mice, with MMP-9 also reduced in ODE-exposed Acod1 -/- mice. Lung function assessments demonstrated a blunted response to LPS-induced airway hyperresponsiveness in Acod1 -/- animals., Conclusion: Acod1 is robustly upregulated in the lungs following LPS exposure and encodes a key immunometabolic regulator. ACOD1 mediates the proinflammatory response to acute inhaled environmental LPS and organic dust exposure-induced lung inflammation., Competing Interests: JP has received research reagent from AstraZeneca (no monies) and has been a site investigator for allergy and asthma clinical studies for Takeda, GlaxoSmithKline, Regeneron, Areteia, and AstraZeneca (no monies). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Schwab, Nelson, Gleason, Schanze, Wyatt, Shinde, Xiao, Thomas, Guda, Bailey, Kielian, Thiele and Poole.)

Background: Craniotomy is a common neurosurgery used to treat intracranial pathologies. Nearly 5% of the 14 million craniotomies performed worldwide each year become infected, most often with Staphylococcus aureus (S. aureus), which forms a biofilm on the surface of the resected bone segment to establish a chronic infection that is recalcitrant to antibiotics and immune-mediated clearance. Tumor necrosis factor (TNF), a prototypical proinflammatory cytokine, has been implicated in generating protective immunity to various infections. Although TNF is elevated during S. aureus craniotomy infection, its functional importance in regulating disease pathogenesis has not been explored., Methods: A mouse model of S. aureus craniotomy infection was used to investigate the functional importance of TNF signaling using TNF, TNFR1, and TNFR2 knockout (KO) mice by quantifying bacterial burden, immune infiltrates, inflammatory mediators, and transcriptional changes by RNA-seq. Complementary experiments examined neutrophil extracellular trap formation, leukocyte apoptosis, phagocytosis, and bactericidal activity., Results: TNF transiently regulated neutrophil and granulocytic myeloid-derived suppressor cell recruitment to the brain, subcutaneous galea, and bone flap as evident by significant reductions in both cell types between days 7 to 14 post-infection coinciding with significant decreases in several chemokines, which recovered to wild type levels by day 28. Despite these defects, bacterial burdens were similar in TNF KO and WT mice. RNA-seq revealed enhanced lymphotoxin-α (Lta) expression in TNF KO granulocytes. Since both TNF and LTα signal through TNFR1 and TNFR2, KO mice for each receptor were examined to assess potential redundancy; however, neither strain had any impact on S. aureus burden. In vitro studies revealed that TNF loss selectively altered macrophage responses to S. aureus since TNF KO macrophages displayed significant reductions in phagocytosis, apoptosis, IL-6 production, and bactericidal activity in response to live S. aureus, whereas granulocytes were not affected., Conclusion: These findings implicate TNF in modulating granulocyte recruitment during acute craniotomy infection via secondary effects on chemokine production and identify macrophages as a key cellular target of TNF action. However, the lack of changes in bacterial burden in TNF KO animals suggests the involvement of additional signals that dictate S. aureus pathogenesis during craniotomy infection., (© 2024. The Author(s).)

Biofilm formation is an important mechanism of survival and persistence for many bacterial pathogens. These multicellular communities contain subpopulations of cells that display vast metabolic and transcriptional diversity along with high recalcitrance to antibiotics and host immune defenses. Investigating the complex heterogeneity within biofilm has been hindered by the lack of a sensitive and high-throughput method to assess stochastic transcriptional activity and regulation between bacterial subpopulations, which requires single-cell resolution. We have developed an optimized bacterial single-cell RNA sequencing method, BaSSSh-seq, to study Staphylococcus aureus diversity during biofilm growth and transcriptional adaptations following immune cell exposure. We validated the ability of BaSSSh-seq to capture extensive transcriptional heterogeneity during biofilm compared to planktonic growth. Application of new computational tools revealed transcriptional regulatory networks across the heterogeneous biofilm subpopulations and identification of gene sets that were associated with a trajectory from planktonic to biofilm growth. BaSSSh-seq also detected alterations in biofilm metabolism, stress response, and virulence that were tailored to distinct immune cell populations. This work provides an innovative platform to explore biofilm dynamics at single-cell resolution, unlocking the potential for identifying biofilm adaptations to environmental signals and immune pressure.

Introduction: Significant neurologic morbidity is caused by pediatric cerebrospinal fluid (CSF) shunt infections. The underlying mechanisms leading to impaired school performance and increased risk of seizures are unknown, however, a better understanding of these mechanisms may allow us to temper their consequences. Recent evidence has demonstrated important roles for complement proteins in neurodevelopment and neuroinflammation., Methods: We examined complement activation throughout Staphylococcus epidermidis ( S. epidermidis ) central nervous system (CNS) catheter infection. In addition, based on accumulating evidence that C3 plays a role in synaptic pruning in other neuroinflammatory states we determined if C3 and downstream C5 led to alterations in synaptic protein levels. Using our murine model of S. epidermidis catheter infection we quantified levels of the complement components C1q, Factor B, MASP2, C3, and C5 over the course of infection along with bacterial burdens., Results: We found that MASP2 predominated early in catheter infection, but that Factor B was elevated at intermediate time points. Unexpectedly C1q was elevated at late timepoints when bacterial burdens were low or undetectable. Based on these findings and the wealth of information regarding the emerging roles of C1q in the CNS, this suggests functions beyond pathogen elimination during S. epidermidis CNS catheter infection. To identify if C3 impacted synaptic protein levels we performed synaptosome isolation and quantified levels of VGLUT1 and PSD95 as well as pre-, post- and total synaptic puncta in cortical layer V of C3 knockout (KO) and wild type mice. We also used C5 KO and wild type mice to determine if there was any difference in pre-, post- and total synaptic puncta., Discussion: Neither C3 nor C5 impacted synaptic protein abundance. These findings suggest that chronic elevations in C1q in the brain that persist once CNS catheter infection has resolved may be modulating disease sequalae., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Beaver, Bergdolt, Dunaevsky, Kielian and Skar.)

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.

Staphylococcus aureus is a leading cause of medical device-associated biofilm infections. This is influenced by the ability of S. aureus biofilm to evade the host immune response, which is partially driven by the anti-inflammatory cytokine interleukin-10 (IL-10). Here, we show that treatment of human monocyte-derived macrophages (HMDMs) with IL-10 enhanced biofilm formation, suggesting that macrophage anti-inflammatory programming likely plays an important role during the transition from planktonic to biofilm growth. To identify S. aureus genes that were important for intracellular survival in HMDMs and how this was affected by IL-10, transposon sequencing was performed. The size of the S. aureus essential genome was similar between unstimulated HMDMs and the outgrowth control (18.5% vs 18.4%, respectively, with 54.4% overlap) but increased to 22.5% in IL-10-treated macrophages, suggesting that macrophage polarization status exerts differential pressure on S. aureus . Essential genes for S. aureus survival within IL-10-polarized HMDMs were dominated by negative regulatory pathways, including nitrogen and RNA metabolism, whereas S. aureus essential genes within untreated HMDMs were enriched in biosynthetic pathways such as purine and pyrimidine biosynthesis. To explore how IL-10 altered the macrophage intracellular metabolome, targeted metabolomics was performed on HMDMs from six individual donors. IL-10 treatment led to conserved alterations in distinct metabolites that were increased (dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, and acetyl-CoA) or reduced (fructose-6-phosphate, aspartic acid, and ornithine) across donors, whereas other metabolites were variable. Collectively, these findings highlight an important aspect of population-level heterogeneity in human macrophage responsiveness that should be considered when translating results to a patient population.IMPORTANCEOne mechanism that Staphylococcus aureus biofilm elicits in the host to facilitate infection persistence is the production of the anti-inflammatory cytokine interleukin-10 (IL-10). Here, we show that exposure of human monocyte-derived macrophages (HMDMs) to IL-10 promotes S. aureus biofilm formation and programs intracellular bacteria to favor catabolic pathways. Examination of intracellular metabolites in HMDMs revealed heterogeneity between donors that may explain the observed variability in essential genes for S. aureus survival based on nutrient availability for bacteria within the intracellular compartment. Collectively, these studies provide novel insights into how IL-10 polarization affects S. aureus intracellular survival in HMDMs and the importance of considering macrophage heterogeneity between human donors as a variable when examining effector mechanisms., Competing Interests: The authors declare no conflict of interest.

Background: Staphylococcus aureus (S. aureus) is a common cause of hospital- and community-acquired infections that can result in various clinical manifestations ranging from mild to severe disease. The bacterium utilizes different combinations of virulence factors and biofilm formation to establish a successful infection, and the emergence of methicillin- and vancomycin-resistant strains introduces additional challenges for infection management and treatment., Summary: Metabolic programming of immune cells regulates the balance of energy requirements for activation and dictates pro- versus anti-inflammatory function. Recent investigations into metabolic adaptations of leukocytes and S. aureus during infection indicate that metabolic crosstalk plays a crucial role in pathogenesis. Furthermore, S. aureus can modify its metabolic profile to fit an array of niches for commensal or invasive growth., Key Messages: Here we focus on the current understanding of immunometabolism during S. aureus infection and explore how metabolic crosstalk between the host and S. aureus influences disease outcome. We also discuss how key metabolic pathways influence leukocyte responses to other bacterial pathogens when information for S. aureus is not available. A better understanding of how S. aureus and leukocytes adapt their metabolic profiles in distinct tissue niches may reveal novel therapeutic targets to prevent or control invasive infections., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

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 (O 2 •- ) from its heme prosthetic group which impedes growth. To limit Hmp toxicity, nitrite (NO 2 - ) 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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 (CX3CR1 Cre IL-10  fl/fl ) or neutrophils and granulocytic myeloid-derived suppressor cells (G-MDSCs; Mrp8 Cre IL-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 Mrp8 Cre IL-10  fl/fl but not CX3CR1 Cre IL-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., (© 2023. The Author(s).)

Background: Cerebrospinal fluid (CSF) shunt infection is a common and devastating complication of the treatment of hydrocephalus. Timely and accurate diagnosis is essential as these infections can lead to long-term neurologic consequences including seizures, decreased intelligence quotient (IQ) and impaired school performance in children. Currently the diagnosis of shunt infection relies on bacterial culture; however, culture is not always accurate since these infections are frequently caused by bacteria capable of forming biofilms, such as Staphylococcus epidermidis, Cutibacterium acnes , and Pseudomonas aeruginosa resulting in few planktonic bacteria detectable in the CSF. Therefore, there is a critical need to identify a new rapid, and accurate method for diagnosis of CSF shunt infection with broad bacterial species coverage to improve the long-term outcomes of children suffering from these infections., Methods: To investigate potential biomarkers that would discriminate S. epidermidis, C. acnes and P. aeruginosa central nervous system (CNS) catheter infection we leveraged our previously published rat model of CNS catheter infection to perform serial CSF sampling to characterize the CSF proteome during these infections compared to sterile catheter placement., Results: P. aeruginosa infection demonstrated a far greater number of differentially expressed proteins when compared to S. epidermidis and C. acnes infection and sterile catheters, and these changes persisted throughout the 56-day time course. S. epidermidis demonstrated an intermediate number of differentially expressed proteins, primarily at early time points that dissipated over the course of infection. C. acnes induced the least amount of change in the CSF proteome when compared to the other pathogens., Conclusions: Despite the differences in the CSF proteome with each organism compared to sterile injury, several proteins were common across all bacterial species, especially at day 5 post-infection, which are candidate diagnostic biomarkers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Beaver, Noe, Thapa, Ali, Snowden, Kielian and Skar.)

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 (Δ pflB ) did not exhibit growth defects in vitro . However, Δ pflB 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 Δ pflB during later stages of infection, which coincided with decreased G-MDSC influx and increased neutrophil recruitment, and Δ pflB was more susceptible to macrophage killing. Although formate was significantly reduced in the soft tissue surrounding the joint of Δ pflB -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 Δ pflB Δ fdh strain defective in formate production and catabolism displayed a similar phenotype to Δ pflB . These results revealed that S. aureus formate metabolism is important for promoting biofilm persistence.

The blood-brain barrier (BBB) selectively restricts the entry of molecules from peripheral circulation into the central nervous system (CNS) parenchyma. Despite this protective barrier, bacteria and other pathogens can still invade the CNS, often as a consequence of immune deficiencies or complications following neurosurgical procedures. These infections are difficult to treat since many bacteria, such as Staphylococcus aureus , encode a repertoire of virulence factors, can acquire antibiotic resistance, and form biofilm. Additionally, pathogens can leverage virulence factor production to polarize host immune cells towards an anti-inflammatory phenotype, leading to chronic infection. The difficulty of pathogen clearance is magnified by the fact that antibiotics and other treatments cannot easily penetrate the BBB, which requires extended regimens to achieve therapeutic concentrations. Nanoparticle systems are rapidly emerging as a promising platform to treat a range of CNS disorders. Nanoparticles have several advantages, as they can be engineered to cross the BBB with specific functionality to increase cellular and molecular targeting, have controlled release of therapeutic agents, and superior bioavailability and circulation compared to traditional therapies. Within the CNS environment, therapeutic actions are not limited to directly targeting the pathogen, but can also be tailored to modulate immune cell activation to promote infection resolution. This perspective highlights the factors leading to infection persistence in the CNS and discusses how novel nanoparticle therapies can be engineered to provide enhanced treatment, specifically through modulation of immune cell polarization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Korshoj, Shi, Duan and Kielian.)

Cutibacterium acnes is the third most common cause of cerebrospinal fluid (CSF) shunt infection and is likely underdiagnosed due to the difficulty in culturing this pathogen. Shunt infections lead to grave neurologic morbidity for patients especially when there is a delay in diagnosis. Currently, the gold standard for identifying CSF shunt infections is microbiologic culture. However, C. acnes infection often results in falsely negative cultures; therefore, new diagnostic methods are needed. To investigate potential CSF biomarkers of C. acnes CSF shunt infection we adapted a rat model of CSF catheter infection to C. acnes. We found elevated levels of interleukin-1β (IL-1β), IL-6, chemokine ligand 2, and IL-10 in the CSF and brain tissues of animals implanted with C. acnes- infected catheters compared to sterile controls at day 1 postinfection. This coincided with modest increases in neutrophils in the CSF and, to a greater extent, in the brain tissues of animals with C. acnes infection, which closely mirrors the clinical findings in patients with C. acnes shunt infection. Mass spectrometry revealed that the CSF proteome is altered during C. acnes shunt infection and changes over the course of disease, typified at day 1 postinfection by an acute-phase and pathogen neutralization response evolving to a response consistent with wound resolution at day 28 compared to a sterile catheter placement. Collectively, these results demonstrate that it is possible to distinguish C. acnes infection from sterile postoperative inflammation and that CSF proteins could be useful in a diagnostic strategy for this pathogen that is difficult to diagnose., (Copyright © 2021 American Society for Microbiology.)

Bacterial infections in the central nervous system (CNS) can be life threatening and often impair neurological function. Biofilm infection is a complication following craniotomy, a neurosurgical procedure that involves the removal and replacement of a skull fragment (bone flap) to access the brain for surgical intervention. The incidence of infection following craniotomy ranges from 1% to 3% with approximately half caused by Staphylococcus aureus ( S. aureus ). These infections present a significant therapeutic challenge due to the antibiotic tolerance of biofilm and unique immune properties of the CNS. Previous studies have revealed a critical role for innate immune responses during S. aureus craniotomy infection. Experiments using knockout mouse models have highlighted the importance of the pattern recognition receptor Toll-like receptor 2 (TLR2) and its adaptor protein MyD88 for preventing S. aureus outgrowth during craniotomy biofilm infection. However, neither molecule affected bacterial burden in a mouse model of S. aureus brain abscess highlighting the distinctions between immune regulation of biofilm vs. planktonic infection in the CNS. Furthermore, the immune responses elicited during S. aureus craniotomy infection are distinct from biofilm infection in the periphery, emphasizing the critical role for niche-specific factors in dictating S. aureus biofilm-leukocyte crosstalk. In this review, we discuss the current knowledge concerning innate immunity to S. aureus craniotomy biofilm infection, compare this to S. aureus biofilm infection in the periphery, and discuss the importance of anatomical location in dictating how biofilm influences inflammatory responses and its impact on bacterial clearance., Competing Interests: A patent has been filed with the US Patent and Trademark Office covering the application of 3D bioprinted scaffolds for the treatment of craniotomy-associated infections that is discussed in this review (PCT/US2020/021440; TK). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 de Morais, Kak, Menousek and Kielian.)

Staphylococcus aureus is a leading cause of bacterial infections globally in both healthcare and community settings. The success of this bacterium is the product of an expansive repertoire of virulence factors in combination with acquired antibiotic resistance and propensity for biofilm formation. S. aureus leverages these factors to adapt to and subvert the host immune response. With the burgeoning field of immunometabolism, it has become clear that the metabolic program of leukocytes dictates their inflammatory status and overall effectiveness in clearing an infection. The metabolic flexibility of S. aureus offers an inherent means by which the pathogen could manipulate the infection milieu to promote its survival. The exact metabolic pathways that S. aureus influences in leukocytes are not entirely understood, and more work is needed to understand how S. aureus co-opts leukocyte metabolism to gain an advantage. In this review, we discuss the current knowledge concerning how metabolic biases dictate the pro- vs. anti-inflammatory attributes of various innate immune populations, how S. aureus metabolism influences leukocyte activation, and compare this with other bacterial pathogens. A better understanding of the metabolic crosstalk between S. aureus and leukocytes may unveil novel therapeutic strategies to combat these devastating infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Horn and Kielian.)

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 (Δ atpA ) 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 Δ atpA 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 Δ atpA biofilm as determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The enhanced inflammatory response elicited by Δ atpA 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, Δ atpA -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 (Δ atpA ) 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 Δ atpA to augment host proinflammatory responses was cell lysis-dependent, as inhibition of bacterial lysis by polyanethole sodium sulfanate or a Δ atpA Δ atl 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., (Copyright © 2020 Bosch et al.)

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-1β, 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.

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.

Background: Environmental organic dust exposures enriched in Toll-like receptor (TLR) agonists can reduce allergic asthma development but are associated with occupational asthma and chronic bronchitis. The TLR adaptor protein myeloid differentiation factor88 (MyD88) is fundamental in regulating acute inflammatory responses to organic dust extract (ODE), yet its role in repetitive exposures is unknown and could inform future strategies., Methods: Wild-type (WT) and MyD88 knockout (KO) mice were exposed intranasally to ODE or saline daily for 3 weeks (repetitive exposure). Repetitively exposed animals were also subsequently rested with no treatments for 4 weeks followed by single rechallenge with saline/ODE., Results: Repetitive ODE exposure induced neutrophil influx and release of pro-inflammatory cytokines and chemokines were profoundly reduced in MyD88 KO mice. In comparison, ODE-induced cellular aggregates, B cells, mast cell infiltrates and serum IgE levels remained elevated in KO mice and mucous cell metaplasia was increased. Expression of ODE-induced tight junction protein(s) was also MyD88-dependent. Following recovery and then rechallenge with ODE, inflammatory mediators, but not neutrophil influx, was reduced in WT mice pretreated with ODE coincident with increased expression of IL-33 and IL-10, suggesting an adaptation response. Repetitively exposed MyD88 KO mice lacked inflammatory responsiveness upon ODE rechallenge., Conclusions: MyD88 is essential in mediating the classic airway inflammatory response to repetitive ODE, but targeting MyD88 does not reduce mucous cell metaplasia, lymphocyte influx, or IgE responsiveness. TLR-enriched dust exposures induce a prolonged adaptation response that is largely MyD88-independent. These findings demonstrate the complex role of MyD88-dependent signaling during acute vs. chronic organic dust exposures.

Implanted medical device-associated infections pose significant health risks, as they are often the result of bacterial biofilm formation. Staphylococcus aureus is a leading cause of biofilm-associated infections which persist due to mechanisms of device surface adhesion, biofilm accumulation, and reprogramming of host innate immune responses. We found that the S. aureus fibronectin binding protein A (FnBPA) is required for normal biofilm development in mammalian serum and that the SaeRS two-component system is required for functional FnBPA activity in serum. Furthermore, serum-developed biofilms deficient in FnBPA were more susceptible to macrophage invasion, and in a model of biofilm-associated implant infection, we found that FnBPA is crucial for the establishment of infection. Together, these findings show that S. aureus FnBPA plays an important role in physical biofilm development and represents a potential therapeutic target for the prevention and treatment of device-associated infections., (Copyright © 2020 American Society for Microbiology.)

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.

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., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: A provisional patent has been filed with the US Patent and Trademark Office covering the application of nanoparticle targeting of monocyte metabolism for the treatment of biofilm-associated infections (62/730,229; TK, TKB, KJY, XX).

Herein we report the synthesis, SAR, and biological evaluation of a series of 1 H -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., Competing Interests: The authors declare no competing financial interest.

Staphylococcus epidermidis cerebrospinal fluid (CSF) shunt infection is a common complication of hydrocephalus treatment, creating grave neurological consequences for patients, especially when diagnosis is delayed. The current method of diagnosis relies on microbiological culture; however, awaiting culture results may cause treatment delays, or culture may fail to identify infection altogether, so newer methods are needed. To investigate potential CSF biomarkers of S. epidermidis shunt infection, we developed a rat model allowing for serial CSF sampling. We found elevated levels of interleukin-10 (IL-10), IL-1β, chemokine ligand 2 (CCL2), and CCL3 in the CSF of animals implanted with S. epidermidis -infected catheters compared to sterile controls at day 1 postinfection. Along with increased chemokine and cytokine expression early in infection, neutrophil influx was significantly increased in the CSF of animals with infected catheters, suggesting that coupling leukocyte counts with inflammatory mediators may differentiate infection from sterile inflammation. Mass spectrometry analysis revealed that the CSF proteome in sterile animals was similar to that in infected animals at day 1; however, by day 5 postinfection, there was an increase in the number of differently expressed proteins in the CSF of infected compared to sterile groups. The expansion of the proteome at day 5 postinfection was interesting, as bacterial burdens began to decline by this point, yet the CSF proteome data indicated that the host response remained active, especially with regard to the complement cascade. Collectively, these results provide potential biomarkers to distinguish S. epidermidis infection from sterile postoperative inflammation., (Copyright © 2019 Skar et al.)

Staphylococcus aureus has the ability to cause infections in multiple organ systems, suggesting an ability to rapidly adapt to changing carbon and nitrogen sources. Although there is little information about the nutrients available at specific sites of infection, a mature skin abscess has been characterized as glucose depleted, indicating that peptides and free amino acids are an important source of nutrients for the bacteria. Our studies have found that mutations in enzymes necessary for growth on amino acids, including pyruvate carboxykinase (Δ pckA ) and glutamate dehydrogenase (Δ gudB ), reduced the ability of the bacteria to proliferate within a skin abscess, suggesting that peptides and free amino acids are important for S. aureus growth. Furthermore, we found that collagen, an abundant host protein that is present throughout a skin abscess, serves as a reservoir of peptides. To liberate peptides from the collagen, we identified that the host protease, MMP-9, as well as the staphylococcal proteases aureolysin and staphopain B function to cleave collagen into peptide fragments that can support S. aureus growth under nutrient-limited conditions. Moreover, the oligopeptide transporter Opp3 is the primary staphylococcal transporter responsible for peptide acquisition. Lastly, we observed that the presence of peptides (3-mer to 7-mer) induces the expression of aureolysin, suggesting that S. aureus has the ability to detect peptides in the environment. IMPORTANCE Staphylococcus aureus has the ability to cause infections in a variety of niches, suggesting a robust metabolic capacity facilitating proliferation under various nutrient conditions. The mature skin abscess is glucose depleted, indicating that peptides and free amino acids are important sources of nutrients for S. aureus Our studies have found that mutations in both pyruvate carboxykinase and glutamate dehydrogenase, enzymes that function in essential gluconeogenesis reactions when amino acids serve as the major carbon source, reduce bacterial burden in a murine skin abscess model. Moreover, peptides liberated from collagen by host protease MMP-9 as well as the staphylococcal protease aureolysin support S. aureus growth in an Opp3-dependent manner under nutrient-limited conditions. Additionally, the presence of peptides induces aureolysin expression. Overall, these studies define one pathway by which S. aureus senses a nutrient-limiting environment and induces factors that function to acquire and utilize carbon from host-derived sources., (Copyright © 2019 Lehman et al.)

Staphylococcus aureus causes acute and chronic infections resulting in significant morbidity. Urease, an enzyme that generates NH3 and CO2 from urea, is key to pH homeostasis in bacterial pathogens under acidic stress and nitrogen limitation. However, the function of urease in S. aureus niche colonization and nitrogen metabolism has not been extensively studied. We discovered that urease is essential for pH homeostasis and viability in urea-rich environments under weak acid stress. The regulation of urease transcription by CcpA, Agr, and CodY was identified in this study, implying a complex network that controls urease expression in response to changes in metabolic flux. In addition, it was determined that the endogenous urea derived from arginine is not a significant contributor to the intracellular nitrogen pool in non-acidic conditions. Furthermore, we found that during a murine chronic renal infection, urease facilitates S. aureus persistence by promoting bacterial fitness in the low-pH, urea-rich kidney. Overall, our study establishes that urease in S. aureus is not only a primary component of the acid response network but also an important factor required for persistent murine renal infections., Competing Interests: The authors have declared that no competing interests exist.

Biofilms are bacterial communities contained within an extracellular matrix, which can colonize both native tissues and artificial surfaces. In particular, indwelling medical devices and prosthetic implants are targets for biofilm formation because they facilitate bacterial attachment via host proteins that coat the foreign body. Biofilm infections are particularly challenging to treat, since they are not readily cleared by antibiotics, require invasive procedures to eradicate, and are prone to recurrence. It has been demonstrated that biofilm-derived products can actively suppress proinflammatory immune responses, as evident by the recruitment of myeloid-derived suppressor cells and macrophage (MФ) polarization towards an anti-inflammatory state. Recent studies have shown that alterations in leukocyte metabolism shape their inflammatory phenotype and function. For example, anti-inflammatory MФs are biased towards oxidative phosphorylation whereas proinflammatory MФs favor aerobic glycolysis. This review will compare the immune responses elicited by planktonic and biofilm bacterial infections, with a discussion on the metabolic properties of MФs and neutrophils in response to both bacterial growth conditions., (The Author(s). Published by S. Karger AG, Basel.)

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 Staphylococcus aureus 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, CD11b high Ly6G + Ly6C + MDSCs and CD11b low Ly6G + Ly6C + PMNs, which was confirmed by characteristic nuclear morphology using cytospins. CD11b high Ly6G + MDSCs suppressed T cell proliferation throughout the 28-day infection period, whereas CD11b + MDSCs suppressed T cell proliferation throughout the 28-day infection period, whereas CD11b low 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)., (Copyright © 2018 American Society for Microbiology.)

CLN3-Batten disease is a rare, autosomal recessive disorder involving seizures, visual, motor and cognitive decline, and premature death. The Cln3Δex7/8 mouse model recapitulates several phenotypic characteristics of the most common 1.02kb disease-associated deletion. Identification of reproducible biomarker(s) to facilitate longitudinal monitoring of disease progression and provide readouts for therapeutic response has remained elusive. One factor that has complicated the identification of suitable biomarkers in this mouse model has been that variations in animal husbandry appear to significantly influence readouts. In the current study, we cross-compared a number of biological parameters in blood from Cln3Δex7/8 mice and control, non-disease mice on the same genetic background from multiple animal facilities in an attempt to better define a surrogate marker of CLN3-Batten disease. Interestingly, we found that significant differences between Batten and non-disease mice found at one site were generally not maintained across different facilities. Our results suggest that colony variation in the Cln3Δex7/8 mouse model of CLN3-Batten disease can influence potential biomarkers of the disease., Competing Interests: The authors have declared that no competing interests exist.

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-2 Cre mice. Despite Arg-1 expression in biofilm-associated myeloid cells, bacterial burdens and leukocyte infiltrates were similar between wild-type (WT) and Arg-1 fl/fl ; Tie-2 Cre 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-1 fl/fl ; Tie-2 Cre animals, suggesting a potential Arg-1-independent compensatory mechanism for MDSC-mediated immunomodulation. Treatment of Arg-1 fl/fl ; Tie-2 Cre 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-1 fl/fl ; Tie-2 Cre 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., (Copyright © 2018 American Society for Microbiology.)

Biofilms are multicellular communities of microorganisms living as a quorum rather than as individual cells. The bacterial human pathogen Staphylococcus aureus uses oxygen as a terminal electron acceptor during respiration. Infected human tissues are hypoxic or anoxic. We recently reported that impaired respiration elicits a p rogrammed c ell l ysis (PCL) phenomenon in S. aureus leading to the release of cellular polymers that are utilized to form biofilms. PCL is dependent upon the AtlA murein hydrolase and is regulated, in part, by the SrrAB two-component regulatory system (TCRS). In the current study, we report that the SaeRS TCRS also governs fermentative biofilm formation by positively influencing AtlA activity. The SaeRS-modulated factor fibronectin-binding protein A (FnBPA) also contributed to the fermentative biofilm formation phenotype. SaeRS-dependent biofilm formation occurred in response to changes in cellular respiratory status. Genetic evidence presented suggests that a high cellular titer of phosphorylated SaeR is required for biofilm formation. Epistasis analyses found that SaeRS and SrrAB influence biofilm formation independently of one another. Analyses using a mouse model of orthopedic implant-associated biofilm formation found that both SaeRS and SrrAB govern host colonization. Of these two TCRSs, SrrAB was the dominant system driving biofilm formation in vivo We propose a model wherein impaired cellular respiration stimulates SaeRS via an as yet undefined signal molecule(s), resulting in increasing expression of AtlA and FnBPA and biofilm formation., (Copyright © 2017 American Society for Microbiology.)

Staphylococcus aureus is a leading cause of community- and nosocomial-acquired infections, with a propensity for biofilm formation. S. aureus biofilms actively skew the host immune response toward an anti-inflammatory state; however, the biofilm effector molecules and the mechanism(s) of action responsible for this phenomenon remain to be fully defined. The essential bacterial second messenger cyclic diadenylate monophosphate (c-di-AMP) is an emerging pathogen-associated molecular pattern during intracellular bacterial infections, as c-di-AMP secretion into the infected host cytosol induces a robust type I interferon (IFN) response. Type I IFNs have the potential to exacerbate infectious outcomes by promoting anti-inflammatory effects; however, the type I IFN response to S. aureus biofilms is unknown. Additionally, while several intracellular proteins function as c-di-AMP receptors in S. aureus, it has yet to be determined if any extracellular role for c-di-AMP exists and its release during biofilm formation has not yet been demonstrated. This study examined the possibility that c-di-AMP released during S. aureus biofilm growth polarizes macrophages toward an anti-inflammatory phenotype via type I interferon signaling. DacA, the enzyme responsible for c-di-AMP synthesis in S. aureus, was highly expressed during biofilm growth, and 30 to 50% of total c-di-AMP produced from S. aureus biofilm was released extracellularly due to autolytic activity. S. aureus biofilm c-di-AMP release induced macrophage type I IFN expression via a STING-dependent pathway and promoted S. aureus intracellular survival in macrophages. These findings identify c-di-AMP as another mechanism for how S. aureus biofilms promote macrophage anti-inflammatory activity, which likely contributes to biofilm persistence., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Clinical post-influenza Staphylococcus aureus pneumonia is characterized by extensive lung inflammation associated with severe morbidity and mortality even after appropriate antibiotic treatment. In this study, we show that antibiotics rescue nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (Nox2)-deficient mice but fail to fully protect WT animals from influenza and S. aureus coinfection. Further experiments indicate that the inefficacy of antibiotics against coinfection is attributable to oxidative stress-associated inflammatory lung injury. However, Nox2-induced lung damage during coinfection was not associated with aggravated inflammatory cytokine response or cell infiltration but rather caused by reduced survival of myeloid cells. Specifically, oxidative stress increased necrotic death of inflammatory cells, thereby resulting in lethal damage to surrounding tissue. Collectively, our results demonstrate that influenza infection disrupts the delicate balance between Nox2-dependent antibacterial immunity and inflammation. This disruption leads to not only increased susceptibility to S. aureus infection, but also extensive lung damage. Importantly, we show that combination treatment of antibiotic and NADPH oxidase inhibitor significantly improved animal survival from coinfection. These findings suggest that treatment strategies that target both bacteria and oxidative stress will significantly benefit patients with influenza-complicated S. aureus pneumonia., (© 2016 Sun et al.)

Staphylococcus aureus is a leading cause of prosthetic joint infections (PJIs) that are typified by biofilm formation. Given the diversity of S. aureus strains and their propensity to cause community- or hospital-acquired infections, we investigated whether the immune response and biofilm growth during PJI were conserved among distinct S. aureus clinical isolates. Three S. aureus strains representing USA200 (UAMS-1), USA300 (LAC), and USA400 (MW2) lineages were equally effective at biofilm formation in a mouse model of PJI and elicited similar leukocyte infiltrates and cytokine/chemokine profiles. Another factor that may influence the course of PJI is infectious dose. In particular, higher bacterial inocula could accelerate biofilm formation and alter the immune response, making it difficult to discern underlying pathophysiological mechanisms. To address this issue, we compared the effects of two bacterial doses (10(3) or 10(5) CFU) on inflammatory responses in interleukin-12p40 (IL-12p40) knockout mice that were previously shown to have reduced myeloid-derived suppressor cell recruitment concomitant with bacterial clearance after low-dose challenge (10(3) CFU). Increasing the infectious dose of LAC to 10(5) CFU negated these differences in IL-12p40 knockout animals, demonstrating the importance of bacterial inoculum on infection outcome. Collectively, these observations highlight the importance of considering infectious dose when assessing immune responsiveness, whereas biofilm formation during PJI is conserved among clinical isolates commonly used in mouse S. aureus infection models., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The Neuronal Ceroid Lipofuscinoses (NCLs) are a family of autosomal recessive neurodegenerative disorders that annually affect 1:100,000 live births worldwide. This family of diseases results from mutations in one of 14 different genes that share common clinical and pathological etiologies. Clinically, the diseases are subcategorized into infantile, late-infantile, juvenile and adult forms based on their age of onset. Though the disease phenotypes may vary in their age and order of presentation, all typically include progressive visual deterioration and blindness, cognitive impairment, motor deficits and seizures. Pathological hallmarks of NCLs include the accumulation of storage material or ceroid in the lysosome, progressive neuronal degeneration and massive glial activation. Advances have been made in genetic diagnosis and counseling for families. However, comprehensive treatment programs that delay or halt disease progression have been elusive. Current disease management is primarily targeted at controlling the symptoms rather than "curing" the disease. Recognizing the growing need for transparency and synergistic efforts to move the field forward, this review will provide an overview of the therapeutic approaches currently being pursued in preclinical and clinical trials to treat different forms of NCL as well as provide insight to novel therapeutic approaches in development for the NCLs.

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., (© Society for Leukocyte Biology.)

Lysosomal storage diseases (LSDs) include approximately 70 distinct disorders that collectively account for 14% of all inherited metabolic diseases. LSDs are caused by mutations in various enzymes/proteins that disrupt lysosomal function, which impairs macromolecule degradation following endosome-lysosome and phagosome-lysosome fusion and autophagy, ultimately disrupting cellular homeostasis. LSDs are pathologically typified by lysosomal inclusions composed of a heterogeneous mixture of various proteins and lipids that can be found throughout the body. However, in many cases the CNS is dramatically affected, which may result from heightened neuronal vulnerability based on their post-mitotic state. Besides intrinsic neuronal defects, another emerging factor common to many LSDs is neuroinflammation, which may negatively impact neuronal survival and contribute to neurodegeneration. Microglial and astrocyte activation is a hallmark of many LSDs that affect the CNS, which often precedes and predicts regions where eventual neuron loss will occur. However, the timing, intensity, and duration of neuroinflammation may ultimately dictate the impact on CNS homeostasis. For example, a transient inflammatory response following CNS insult/injury can be neuroprotective, as glial cells attempt to remove the insult and provide trophic support to neurons. However, chronic inflammation, as seen in several LSDs, can promote neurodegeneration by creating a neurotoxic environment due to elevated levels of cytokines, chemokines, and pro-apoptotic molecules. Although neuroinflammation has been reported in several LSDs, the cellular basis and mechanisms responsible for eliciting neuroinflammatory pathways are just beginning to be defined. This review highlights the role of neuroinflammation in select LSDs and its potential contribution to neuron loss.

Inhalation of organic dusts within agriculture environments contributes to the development and/or severity of airway diseases, including asthma and chronic bronchitis. MyD88 KO (knockout) mice are nearly completely protected against the inflammatory and bronchoconstriction effects induced by acute organic dust extract (ODE) treatments. However, the contribution of MyD88 in lung epithelial cell responses remains unclear. In the present study, we first addressed whether ODE-induced changes in epithelial cell responses were MyD88-dependent by quantitating ciliary beat frequency and cell migration following wounding by electric cell-substrate impedance sensing. We demonstrate that the normative ciliary beat slowing response to ODE is delayed in MyD88 KO tracheal epithelial cells as compared to wild type (WT) control. Similarly, the normative ODE-induced slowing of cell migration in response to wound repair was aberrant in MyD88 KO cells. Next, we created MyD88 bone marrow chimera mice to investigate the relative contribution of MyD88-dependent signaling in lung resident (predominately epithelial cells) versus hematopoietic cells. Importantly, we demonstrate that ODE-induced airway hyperresponsiveness is MyD88-dependent in lung resident cells, whereas MyD88 action in hematopoietic cells is mainly responsible for ODE-induced TNF-α release. MyD88 signaling in lung resident and hematopoietic cells are necessary for ODE-induced IL-6 and neutrophil chemoattractant (CXCL1 and CXCL2) release and neutrophil influx. Collectively, these findings underscore an important role for MyD88 in lung resident cells for regulating ciliary motility, wound repair and inflammatory responses to ODE, and moreover, show that airway hyperresponsiveness appears uncoupled from airway inflammatory consequences to organic dust challenge in terms of MyD88 involvement.

Unlabelled: The macrophage response to planktonic Staphylococcus aureus involves the induction of proinflammatory microbicidal activity. However, S. aureus biofilms can interfere with these responses in part by polarizing macrophages toward an anti-inflammatory profibrotic phenotype. Here we demonstrate that conditioned medium from mature S. aureus biofilms inhibited macrophage phagocytosis and induced cytotoxicity, suggesting the involvement of a secreted factor(s). Iterative testing found the active factor(s) to be proteinaceous and partially agr-dependent. Quantitative mass spectrometry identified alpha-toxin (Hla) and leukocidin AB (LukAB) as critical molecules secreted by S. aureus biofilms that inhibit murine macrophage phagocytosis and promote cytotoxicity. A role for Hla and LukAB was confirmed by using hla and lukAB mutants, and synergy between the two toxins was demonstrated with a lukAB hla double mutant and verified by complementation. Independent confirmation of the effects of Hla and LukAB on macrophage dysfunction was demonstrated by using an isogenic strain in which Hla was constitutively expressed, an Hla antibody to block toxin activity, and purified LukAB peptide. The importance of Hla and LukAB during S. aureus biofilm formation in vivo was assessed by using a murine orthopedic implant biofilm infection model in which the lukAB hla double mutant displayed significantly lower bacterial burdens and more macrophage infiltrates than each single mutant. Collectively, these findings reveal a critical synergistic role for Hla and LukAB in promoting macrophage dysfunction and facilitating S. aureus biofilm development in vivo., Importance: Staphylococcus aureus has a propensity to form multicellular communities known as biofilms. While growing in a biofilm, S. aureus displays increased tolerance to nutrient deprivation, antibiotic insult, and even host immune challenge. Previous studies have shown that S. aureus biofilms thwart host immunity in part by preventing macrophage phagocytosis. It remained unclear whether this was influenced solely by the considerable size of biofilms or whether molecules were also actively secreted to circumvent macrophage-mediated phagocytosis. This is the first report to demonstrate that S. aureus biofilms inhibit macrophage phagocytosis and induce macrophage death through the combined action of leukocidin AB and alpha-toxin. Loss of leukocidin AB and alpha-toxin expression resulted in enhanced S. aureus biofilm clearance in a mouse model of orthopedic implant infection, suggesting that these toxins could be targeted therapeutically to facilitate biofilm clearance in humans., (Copyright © 2015 Scherr et al.)

Staphylococcus aureus is a significant cause of chronic biofilm infections on medical implants. We investigated the biofilm regulatory cascade and discovered that the repressor of toxins (Rot) is part of this pathway. A USA300 community-associated methicillin-resistant S. aureus strain deficient in Rot was unable to form a biofilm using multiple different assays, and we found rot mutants in other strain lineages were also biofilm deficient. By performing a global analysis of transcripts and protein production controlled by Rot, we observed that all the secreted protease genes were up-regulated in a rot mutant, and we hypothesized that this regulation could be responsible for the biofilm phenotype. To investigate this question, we determined that Rot bound to the protease promoters, and we observed that activity levels of these enzymes, in particular the cysteine proteases, were increased in a rot mutant. By inactivating these proteases, biofilm capacity was restored to the mutant, demonstrating they are responsible for the biofilm negative phenotype. Finally, we tested the rot mutant in a mouse catheter model of biofilm infection and observed a significant reduction in biofilm burden. Thus S. aureus uses the transcription factor Rot to repress secreted protease levels in order to build a biofilm., (© 2015 John Wiley & Sons Ltd.)

Background: Laryngeal injury associated with traumatic or prolonged intubation may lead to voice, swallow, and airway complications. The interplay between inflammation and microbial population shifts induced by intubation may relate to clinical outcomes. The objective of this study was to investigate laryngeal mechanics, tissue inflammatory response, and local microbiome changes with laryngotracheal injury and localized delivery of therapeutics via drug-eluting endotracheal tube. Methods: A simulated traumatic intubation injury was created in Yorkshire crossbreed swine under direct laryngoscopy. Endotracheal tubes electrospun with roxadustat or valacyclovir- loaded polycaprolactone (PCL) fibers were placed in the injured airway for 3, 7, or 14 days (n = 3 per group/time and ETT type). Vocal fold stiffness was then evaluated with normal indentation and laryngeal tissue sections were histologically examined. Immunohistochemistry and inflammatory marker profiling were conducted to evaluate the inflammatory response associated with injury and ETT placement. Additionally, ETT biofilm formation was visualized using scanning electron microscopy and micro-computed tomography, while changes in the airway microbiome were profiled through 16S rRNA sequencing. Results: Laryngeal tissue with roxadustat ETT placement had increasing localized stiffness outcomes over time and histological assessment indicated minimal epithelial ulceration and fibrosis, while inflammation remained severe across all timepoints. In contrast, vocal fold tissue with valacyclovir ETT placement showed no significant changes in stiffness over time; histological analysis presented a reduction in epithelial ulceration and inflammation scores along with increased fibrosis observed at 14 days. Immunohistochemistry revealed a decline in M1 and M2 macrophage markers over time for both ETT types. Among the cytokines, IL-8 levels differed significantly between the roxadustat and valacyclovir ETT groups, while no other cytokines showed statistically significant differences. Additionally, increased biofilm formation was observed in the coated ETTs with notable alterations in microbiota distinctive to each ETT type and across time. Conclusion: The injured and intubated airway resulted in increased laryngeal stiffness. Local inflammation and the type of therapeutic administered impacted the bacterial composition within the upper respiratory microbiome, which in turn mediated local tissue healing and recovery. [ABSTRACT FROM AUTHOR]

The conditions supporting the generation of microglia-like cells in the central nervous system (CNS) after transplantation of hematopoietic stem/progenitor cells (HSPC) have been studied to advance the treatment of neurodegenerative disorders. Here, we explored the transplantation efficacy of different cell subsets and delivery routes with the goal of favoring the establishment of a stable and exclusive engraftment of HSPCs and their progeny in the CNS of female mice. In this setting, we show that the CNS environment drives the expansion, distribution and myeloid differentiation of the locally transplanted cells towards a microglia-like phenotype. Intra-CNS transplantation of HSPCs engineered to overexpress TREM2 decreased neuroinflammation, Aβ aggregation and improved memory in 5xFAD female mice. Our proof of concept study demonstrates the therapeutic potential of HSPC gene therapy for Alzheimer's disease. This study aims at enhancing hematopoietic stem cell (HSC) gene therapy potential to treat neurodegenerative diseases via innovative approaches. Findings demonstrate the therapeutic benefits of intracerebral transplantation of TREM2-engineered HSCs in an Alzheimer's disease mouse model. [ABSTRACT FROM AUTHOR]

Background and Objective: This randomized controlled trial investigated the influence of perioperative lidocaine administration on the postoperative inflammatory response in patients undergoing robot-assisted radical prostatectomy, with the results having potential implications for postoperative recovery and cancer recurrence via neutrophil extracellular trapping (NETosis). Materials and Methods: In total, 58 patients with localized prostate cancer were randomly assigned to receive an intravenous infusion of 2% lidocaine or a saline placebo intraoperatively. Serum levels of interleukin (IL)-6, IL-10, and IL-17, tumor necrosis factor(TNF)-α, interferon(IFN)-γ, neutrophil elastase (NE), citrullinated histone3 (CitH3), and myeloperoxidase (MPO) were determined preoperatively and at 24 h postoperatively. Biochemical recurrence (BCR) was assessed over a follow-up period of 2 years. Results: The lidocaine group showed a significant change in MPO, a greater reduction in IL-10 level, and a smaller increase in the NE level compared to the placebo group, suggesting a modulatory effect of lidocaine on certain anti-inflammatory and neuroendocrine pathways. No significant difference in the BCR rate was observed between the two groups. Conclusions: Perioperative lidocaine administration selectively modulates certain inflammatory and neuroendocrine responses after robot-assisted radical prostatectomy surgery, potentially influencing recovery outcomes. These findings highlight the need for further investigations of the role of lidocaine in Enhanced Recovery After Surgery protocols, particularly in oncologic surgeries. [ABSTRACT FROM AUTHOR]