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.

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.

Immunopathogenesis of Craniotomy Infection and Niche-Specific Immune Responses to Biofilm.

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.

Regulation of Interferon-γ receptor (IFN-γR) expression in macrophages during Mycobacterium tuberculosis infection.

Interferon-gamma (IFN-γ) is a key cytokine that mediates immunity to tuberculosis (TB). Mycobacterium tuberculosis (M. tb) is known to downregulate the surface expression of IFN-γ receptor (IFN-γR) on macrophages and peripheral blood mononuclear cells (PBMCs) of patients with active TB disease. Many M. tb antigens also downmodulate IFN-γR levels in macrophages when compared with healthy controls. In the current study, we aimed at deciphering key factors involved in M. tb mediated downregulation of IFN-γR levels on macrophage surface. Our data showed that both M. tb H37Rv and M. bovis BCG infections mediate downmodulation of IFN-γR on human macrophages. This downmodulation is regulated at the level of TLR signaling pathway, second messengers such as calcium and cellular kinases i.e. PKC and ERK-MAPK, indicating that fine tuning of calcium response is critical to maintaining IFN-γR levels on macrophage surface. In addition, genes in the calcium and cysteine protease pathways which were previously identified by us to play a negative role during M. tb infection, also regulated IFN-γR expression. Thus, modulations in IFN-γR levels by utilizing host machinery may be a key immune suppressive strategy adopted by the TB pathogen to ensure its persistence and thwart host defense.

Interferon-gamma (IFN-γ): Exploring its implications in infectious diseases.

A key player in driving cellular immunity, IFN-γ is capable of orchestrating numerous protective functions to heighten immune responses in infections and cancers. It can exhibit its immunomodulatory effects by enhancing antigen processing and presentation, increasing leukocyte trafficking, inducing an anti-viral state, boosting the anti-microbial functions and affecting cellular proliferation and apoptosis. A complex interplay between immune cell activity and IFN-γ through coordinated integration of signals from other pathways involving cytokines and Pattern Recognition Receptors (PRRs) such as Interleukin (IL)-4, TNF-α, Lipopolysaccharide (LPS), Type-I Interferons (IFNS) etc. leads to initiation of a cascade of pro-inflammatory responses. Microarray data has unraveled numerous genes whose transcriptional regulation is influenced by IFN-γ. Consequently, IFN-γ stimulated cells display altered expression of many such target genes which mediate its downstream effector functions. The importance of IFN-γ is further reinforced by the fact that mice possessing disruptions in the IFN-γ gene or its receptor develop extreme susceptibility to infectious diseases and rapidly succumb to them. In this review, we attempt to elucidate the biological functions and physiological importance of this versatile cytokine. The functional implications of its biological activity in several infectious diseases and autoimmune pathologies are also discussed. As a counter strategy, many virulent pathogenic species have devised ways to thwart IFN-γ endowed immune-protection. Thus, IFN-γ mediated host-pathogen interactions are critical for our understanding of disease mechanisms and these aspects also manifest enormous therapeutic importance for the annulment of various infections and autoimmune conditions.

Suppression of Protective Responses upon Activation of L-Type Voltage Gated Calcium Channel in Macrophages during Mycobacterium bovis BCG Infection.

The prevalence of Mycobacterium tuberculosis (M. tb) strains eliciting drug resistance has necessitated the need for understanding the complexities of host pathogen interactions. The regulation of calcium homeostasis by Voltage Gated Calcium Channel (VGCCs) upon M. tb infection has recently assumed importance in this area. We previously showed a suppressor role of VGCC during M. tb infections and recently reported the mechanisms of its regulation by M. tb. Here in this report, we further characterize the role of VGCC in mediating defence responses of macrophages during mycobacterial infection. We report that activation of VGCC during infection synergistically downmodulates the generation of oxidative burst (ROS) by macrophages. This attenuation of ROS is regulated in a manner which is dependent on Toll like Receptor (TLR) and also on the route of calcium influx, Protein Kinase C (PKC) and by Mitogen Activation Protein Kinase (MAPK) pathways. VGCC activation during infection increases cell survival and downmodulates autophagy. Concomitantly, pro-inflammatory responses such as IL-12 and IFN-γ secretion and the levels of their receptors on cell surface are inhibited. Finally, the ability of phagosomes to fuse with lysosomes in M. bovis BCG and M. tb H37Rv infected macrophages is also compromised when VGCC activation occurs during infection. The results point towards a well-orchestrated strategy adopted by mycobacteria to supress protective responses mounted by the host. This begins with the increase in the surface levels of VGCCs by mycobacteria and their antigens by well-controlled and regulated mechanisms. Subsequent activation of the upregulated VGCC following tweaking of calcium levels by molecular sensors in turn mediates suppressor responses and prepare the macrophages for long term persistent infection.