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

VGCC activation and mycobacterial infection synergistically lead to suppression of pro-inflammatory cytokine responses in human macrophages.

<p>Human PBMC derived macrophages (Panel A&B) either were infected with 2 MOI <i>M</i>. <i>bovis</i> BCG or stimulated with 50 nM BAYK8644 or both for 24 h. For Panel A, culture supernatant was processed for the estimation of indicated cytokines and bars represent the amount of cytokine in pg/ml. For Panel B, cells were processed for measuring surface densities of indicated cytokine receptors by FACS and bars represent MFI of indicated groups of three independent experiments (n = 3). The results were analyzed by one way ANOVA followed by Tukey’s post hoc multiple comparison test. The star above the bar represents the P value between unstimulated/uninfected and the corresponding group of that bar in each panel. ns = P>0.05; * = P<u><</u> 0.05; ** = P<u><</u>0.01; *** = P<u><</u>0.001 and **** = P<u><</u>0.0001.</p

Calcium, MAP-ERK and PKC modulate ROS generation during infection and VGCC activation.

<p>PMA stimulated THP1 macrophages were treated with inhibitors to indicated molecules for 1 h followed by either infection with 2 MOI <i>M</i>. <i>bovis</i> BCG or stimulation with 50 nM BAYK8644 or both for 1 h. In all the panels, thin line represents ROS generation in unstimulated/uninfected or control cells. Dotted line represent ROS generation in cells infected with <i>M</i>. <i>bovis</i> BCG (Panel A) or stimulation with BAYK8644 (Panel B) or both (Panel C). The thick line in all the panels represent ROS generation in cells either infected with <i>M</i>. <i>bovis</i> BCG (Panel A) or stimulated with BAYK8644 (Panel B) or both (Panel C) following treatment with inhibitors to indicated molecules. Bar graph below each panel represents MFI of the peak at the higher fluorescence in the figure. Data from one of three independent experiments are shown (n = 3). The star above the bar represents the P value between stimulated/infected cells and the corresponding group of that bar chart in each panel. The results were analyzed by one way ANOVA followed by Tukey’s post hoc multiple comparison test. ns = P > 0.05; * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001 and **** = P ≤ 0.0001.</p

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

<div><p>The prevalence of <i>Mycobacterium tuberculosis</i> (<i>M</i>. <i>tb</i>) 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 <i>M</i>. <i>tb</i> infection has recently assumed importance in this area. We previously showed a suppressor role of VGCC during <i>M</i>. <i>tb</i> infections and recently reported the mechanisms of its regulation by <i>M</i>. <i>tb</i>. 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 <i>M</i>. <i>bovis</i> BCG and <i>M</i>. <i>tb</i> 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.</p></div

Activation of VGCC along with <i>M</i>. <i>bovis BCG</i> infection attenuates ROS in THP-1 macrophages.

<p>PMA stimulated THP1 macrophages were either infected with 2 MOI <i>M</i>. <i>bo</i>vis BCG (BCG) or stimulated with 50 nM BAYK8644 or both for indicated times. 30 min before the incubation period, cells were incubated with 10 μM DCFH-DA. After the incubation period, cells were washed with culture medium and ROS levels were analyzed by flow cytometry. In all the Panels, thin line represents ROS generation in uninfected or unstimulated or control cells; thick line represents ROS generation in infected or stimulated cells as indicated. Bar graphs adjacent to histograms in Panel (A-C) show Mean Fluorescent Intensity (MFI) of the peak at the higher fluorescence in the figure. Data from one of three independent experiments are shown (n = 3). The star above the bars represents the P value between control and the corresponding group of that bar in each panel. The results were analyzed by one way ANOVA followed by Tukey’s post hoc multiple comparison test. * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001 and **** = P ≤ 0.0001.</p

VGCC activation and mycobacterial infection synergistically regulate phagosome-lysosome fusion in macrophages.

<p>PMA treated THP1 macrophages (Panel A) and mouse bone marrow derived macrophages (Panel B) were seeded on the coverslip and washed with RPMI 1640 medium and incubated in OPTIMEM medium with or without BAYK8644 for 1 h followed by infection with FM4-64 labeled <i>M</i>. <i>bovis</i> BCG (panel A) or GFP expressing <i>M</i>. <i>tb</i> H37Rv (Panel B) for 4 h. thirty minutes prior to the end of infection period, cells were incubated with 50nM of Lysotracker Green (for Panel A) or Lysotracker Green (for Panel B). At the end of incubation period cells were washed once with PBS and fixed with 4% paraformaldehyde for 1h. Following through washes, the cover slips were mounted with anti-fade containing DAPI. Confocal microscopy was performed on Leica TCS SP-8 confocal instrument, LAX Version 1.8.1.137. Bar chart in both Panel A and Panel B represents percentage of co-localization as determined by LAS AF Version 2.6.0 build 7266 of Leica Micro Systems CMS GmbH. Bars represent percentage of co-localization of the indicated groups of three independent experiments (n = 3). The stars represent the P value between unstimulated and corresponding stimulated (Bay/Amlodipine) group of that bar in each panel. The results were analyzed by one way ANOVA followed by Tukey’s post hoc multiple comparison test. * = P<u><</u> 0.05; ** = P<u><</u>0.01; *** = P<u><</u>0.001 and **** = P<u><</u>0.0001.</p