Exploring epigenetic reprogramming during central nervous system infection.

Epigenetics involves the study of various modes of adaptable transcriptional regulation, contributing to cell identity, characteristics, and function. During central nervous system (CNS) infection, epigenetic mechanisms can exert pronounced control over the maturation and antimicrobial properties of nearly every immune cell type. Epigenetics is a relatively new field, with the first mention of these marks proposed only a half-century ago and a substantial body of immunological epigenetic research emerging only in the last few decades. Here, we review the best-characterized epigenetic marks and their functions as well as illustrate how various immune cell populations responding to CNS infection utilize these marks to organize their activation state and inflammatory processes. We also discuss the metabolic and clinical implications of epigenetic marks and the rapidly expanding set of tools available to researchers that are enabling elucidation of increasingly detailed genetic regulatory pathways. These considerations paint an intricate picture of inflammatory regulation, where epigenetic marks influence genetic, signaling, and environmental elements to orchestrate a tailored immunological response to the threat at hand, cementing epigenetics as an important player in immunity.

Epigenetic Regulation of Leukocyte Inflammatory Mediator Production Dictates Staphylococcus aureus Craniotomy Infection Outcome.

Staphylococcus aureus is a common cause of surgical-site infections, including those arising after craniotomy, which is performed to access the brain for the treatment of tumors, epilepsy, or hemorrhage. Craniotomy infection is characterized by complex spatial and temporal dynamics of leukocyte recruitment and microglial activation. We recently identified unique transcriptional profiles of these immune populations during S. aureus craniotomy infection. Epigenetic processes allow rapid and reversible control over gene transcription; however, little is known about how epigenetic pathways influence immunity to live S. aureus. An epigenetic compound library screen identified bromodomain and extraterminal domain-containing (BET) proteins and histone deacetylases (HDACs) as critical for regulating TNF, IL-6, IL-10, and CCL2 production by primary mouse microglia, macrophages, neutrophils, and granulocytic myeloid-derived suppressor cells in response to live S. aureus. Class I HDACs (c1HDACs) were increased in these cell types in vitro and in vivo during acute disease in a mouse model of S. aureus craniotomy infection. However, substantial reductions in c1HDACs were observed during chronic infection, highlighting temporal regulation and the importance of the tissue microenvironment for dictating c1HDAC expression. Microparticle delivery of HDAC and BET inhibitors in vivo caused widespread decreases in inflammatory mediator production, which significantly increased bacterial burden in the brain, galea, and bone flap. These findings identify histone acetylation as an important mechanism for regulating cytokine and chemokine production across diverse immune cell lineages that is critical for bacterial containment. Accordingly, aberrant epigenetic regulation may be important for promoting S. aureus persistence during craniotomy infection.

Metabolic diversity of human macrophages: potential influence on Staphylococcus aureus intracellular survival.

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.

Tumor necrosis factor regulates leukocyte recruitment but not bacterial persistence during Staphylococcus aureus craniotomy infection.

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.

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.

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 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.

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.

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.

Cutibacterium acnes Central Nervous System Catheter Infection Induces Long-Term Changes in the Cerebrospinal Fluid Proteome.

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-1ß (IL-1ß), 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.

Neuroimmune metabolism: Uncovering the role of metabolic reprogramming in central nervous system disease.

The rapidly expanding field of immunometabolism has highlighted an intricate association between the metabolic pathways that program cellular pro-inflammatory versus anti-inflammatory activity. This Special Issue on Neuroimmune Metabolism showcases a growing body of work characterizing the metabolic profiles of the major CNS-resident and peripheral immune cell players in neuroinflammation, neurodegeneration, and brain injury. The review articles address the roles of glycolytic, oxidative, and lipid metabolism that are associated with beneficial or detrimental properties in various neurological conditions, address unanswered questions in the field, and discuss promising avenues for future therapeutics. Cover Image for this issue: https://doi.org/10.1111/jnc.15069.

Urease is an essential component of the acid response network of Staphylococcus aureus and is required for a persistent murine kidney infection.

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.

Staphylococcus aureus Fibronectin Binding Protein A Mediates Biofilm Development and Infection.

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.

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.

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.

MyD88 regulates a prolonged adaptation response to environmental dust exposure-induced lung disease.

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.

Identification of Potential Cerebrospinal Fluid Biomarkers To Discriminate between Infection and Sterile Inflammation in a Rat Model of Staphylococcus epidermidis Catheter Infection.

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-1ß, 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.

Lysosomal storage disorders: pathology within the lysosome and beyond.

This Preface introduces the articles of the special issue on "Lysosomal Storage Disorders" in which several recognized experts provide an overview of this research field. Lysosomes were first described in the 1950s and recognized for their role in substrate degradation and recycling. Because lysosomes impact numerous fundamental homeostatic processes, research on lysosomal storage disorders (LSDs) is crucial to advance our understanding of this intriguing organelle. This Special Issue highlights some of the LSDs that impact the central nervous system (CNS) as well as comprehensive overviews of lysosomal biology, CNS metabolism, and sphingolipid biosynthesis and turnover, all of which are critical toward our understanding of normal lysosomal function and how this is perturbed in the context of LSDs. This is the Preface for the special issue "Lysosomal Storage Disorders". Cover Image for this issue: doi: 10.1111/jnc.14496.

Astrocytes in juvenile neuronal ceroid lipofuscinosis (CLN3) display metabolic and calcium signaling abnormalities.

Juvenile neuronal ceroid lipofuscinosis (JNCL) is a lysosomal storage disease caused by autosomal recessive mutations in ceroid lipofuscinosis 3 (CLN3). Children with JNCL experience progressive visual, cognitive, and motor deterioration with a decreased life expectancy (late teens-early 20s). Neuronal loss is thought to occur, in part, via glutamate excitotoxicity; however, little is known about astrocyte glutamate regulation in JNCL. Spontaneous Ca2+ oscillations were reduced in murine Cln3Δex7/8 astrocytes, which were also observed following glutamate or cytokine exposure. Astrocyte glutamate transport is an energy-demanding process and disruptions in metabolic pathways could influence glutamate homeostasis in Cln3Δex7/8 astrocytes. Indeed, basal mitochondrial respiration and ATP production were significantly reduced in Cln3Δex7/8 astrocytes. These changes were not attributable to reduced mitochondria, since mitochondrial DNA levels were similar between wild type and Cln3Δex7/8 astrocytes. Interestingly, despite these functional deficits in Cln3Δex7/8 astrocytes, glutamate transporter expression and glutamate uptake were not dramatically affected. Concurrent with impaired astrocyte metabolism and Ca2+ signaling, murine Cln3Δex7/8 neurons were hyper-responsive to glutamate, as reflected by heightened and prolonged Ca2+ signals. These findings identify intrinsic metabolic and Ca2+ signaling defects in Cln3Δex7/8 astrocytes that may contribute to neuronal dysfunction in CLN3 disease. This article is part of the Special Issue "Lysosomal Storage Disorders".

Caspase 1 activity influences juvenile Batten disease (CLN3) pathogenesis.

Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) is an autosomal recessive lysosomal storage disease caused by loss-of-function mutations in CLN3. Symptoms appear between 5 and 10 years of age, beginning with blindness and seizures, followed by progressive cognitive and motor decline, and premature death. Glial activation and impaired neuronal activity are early signs of pathology in the Cln3Δex7/8 mouse model of JNCL, whereas neuron death occurs much later in the disease process. We previously reported that Cln3Δex7/8 microglia are primed toward a pro-inflammatory phenotype typified by exaggerated caspase 1 inflammasome activation and here we extend those findings to demonstrate heightened caspase activity in the Cln3Δex7/8 mouse brain. Based on the ability of caspase 1 to cleave a large number of substrates that have been implicated in JNCL pathology, we examined the functional implications of caspase 1 inflammasome activity by crossing Cln3Δex7/8 and caspase 1-deficient mice to create Cln3Δex7/8 /Casp-1-/- animals. Caspase 1 deletion influenced motor behavior deficits and astrocyte activation in the context of CLN3 mutation, since both were significantly reversed in Cln3Δex7/8 /Casp-1-/- mice, with phenotypes approaching that of wild-type animals. We also report a progressive age-dependent reduction in whisker length in Cln3Δex7/8 mice that was partially caspase 1-dependent. However, not all CLN3 phenotypes were reversed following caspase 1 deletion, since no significant differences in lysosomal accumulation or microglial activation were observed between Cln3Δex7/8 and Cln3Δex7/8 /Casp-1-/- mice. Although the molecular targets of aberrant caspase 1 activity in the context of CLN3 mutation remain to be identified, our studies suggest that caspase 1 may represent a potential therapeutic target to mitigate some attributes of CLN3 disease. This article is part of the Special Issue "Lysosomal Storage Disorders".