CD1d Selectively Down Regulates the Expression of the Oxidized Phospholipid-Specific E06 IgM Natural Antibody in Ldlr−/− Mice

Natural antibodies (NAbs) are important regulators of tissue homeostasis and inflammation and are thought to have diverse protective roles in a variety of pathological states. E06 is a T15 idiotype IgM NAb exclusively produced by B-1 cells, which recognizes the phosphocholine (PC) head group in oxidized phospholipids on the surface of apoptotic cells and in oxidized LDL (OxLDL), and the PC present on the cell wall of Streptococcus pneumoniae. Here we report that titers of the E06 NAb are selectively increased several-fold in Cd1d-deficient mice, whereas total IgM and IgM antibodies recognizing other oxidation specific epitopes such as in malondialdehyde-modified LDL (MDA-LDL) and OxLDL were not increased. The high titers of E06 in Cd1d-deficient mice are not due to a global increase in IgM-secreting B-1 cells, but they are specifically due to an expansion of E06-secreting splenic B-1 cells. Thus, CD1d-mediated regulation appeared to be suppressive in nature and specific for E06 IgM-secreting cells. The CD1d-mediated regulation of the E06 NAb generation is a novel mechanism that regulates the production of this specific oxidation epitope recognizing NAb.

INNATE IMMUNE INTERFERENCE ATTENUATES INFLAMMATION IN BACILLUS ENDOPHTHALMITIS

ABSTRACTPURPOSEBacillus endophthalmitis is a sight-threatening bacterial infection that sometimes requires enucleation. Inflammation in this disease is driven by activation of innate Toll-like receptor (TLR) pathways. Here, we explored the consequences of innate immune interference on intraocular inflammatory responses during Bacillus endophthalmitis.METHODSEndophthalmitis was induced in mice by injecting 100 CFU Bacillus thuringiensis in to the mid-vitreous. We interfered with activation of the TLR2 and TLR4 pathways by 1) injecting a group of mice with S layer protein-deficient (ΔslpA) B. thuringiensis or 2) injecting a group of wild type (WT)-infected mice with a TLR2/4 inhibitor, oxidized phospholipid (OxPAPC). At 10 hours postinfection, infected eyes were removed and total RNA was purified. mRNA expression was then analyzed by NanoString using a murine inflammation panel. We compared findings with expression data from eyes infected with eyes injected with WT B. thuringiensis, eyes injected with OxPAPC alone, and uninfected eyes.RESULTSInterference of TLR2 and TLR4 pathways resulted in differential expression of mouse inflammatory genes compared to expression in WT-infected eyes. In WT-infected eyes, 56% of genes were significantly upregulated compared to that of uninfected controls. However, compared to WT-infected eyes, the expression of 27% and 50% of genes were significantly reduced in WT+OxPAPC and ΔslpA-infected eyes, respectively. The expression of 61 genes which were significantly upregulated in WT-infected eyes was decreased in WT+OxPAPC or ΔslpA-infected eyes. Interference with activation of the TLR2 and TLR4 pathways resulted in blunted expression of complement factors (C3, Cfb, and C6) and several innate genes such as TLR2, TLR4, TLR6, TLR8, MyD88, Nod2, Nlrp3, NF-κB, STAT3, RelA, RelB, and Ptgs2. Interference with activation of the TLR2 and TLR4 pathways also reduced the expression of several inflammatory cytokines such as CSF3, IL-6, IL-1β, CSF2, IL-1α, TNFα, IL-23α, TGFβ1, and IL-12β and chemokines CCL2, CCl3, CXCL1, CXCL2, CXCL3, CXCL5, CXCL9, and CXCL10. All of the aforementioned genes were significantly upregulated in WT-infected eyes.CONCLUSIONSThese results suggest that interfering with the activation of innate immune pathways during Bacillus endophthalmitis significantly reduced the intraocular inflammatory response. This positive clinical outcome could be a strategy for anti-inflammatory therapy of an infection typically refractory to corticosteroid treatment.

Oxidation of Peroxiredoxin 6 in the Presence of GSH Increases its Phospholipase A2 Activity at Cytoplasmic pH

The expression of the phospholipase A2 activity (aiPLA2) of peroxiredoxin 6 (Prdx6) in the cell cytoplasm is physiologically relevant for the repair of peroxidized cell membranes, but aiPLA2 assay in vitro indicates that, unlike assay at pH 4, activity at cytosolic pH is essentially absent with non-oxidized substrate. However, the addition of glutathione (GSH) to the assay medium significantly increased aiPLA2 activity at cytosolic pH, while oxidized GSH (GSSG) and several other thiols had no effect. By mass spectroscopy (ESI MS), the addition of GSH to Prdx6 paradoxically led to oxidation of its conserved Cys47 residue to a sulfinic acid. The effect of GSH on PLA2 activity was abolished by incubation under anaerobic conditions, confirming that auto-oxidation of the protein was the mechanism for the GSH effect. Analysis by circular dichroism (CD) and tryptophan fluorescence showed alterations of the protein structure in the presence of GSH. Independently of GSH, the oxidation of Prdx6 by exposure to H2O2 or the presence of oxidized phospholipid as substrate also significantly increased aiPLA2 activity at pH 7. We conclude that the oxidation of the peroxidatically active Cys47 of Prdx6 results in an increase of aiPLA2 activity at pH 7 without effect on the activity of the enzyme at pH 4.

Effect of methylene blue on the formation of oxidized phospholipid vesicles

Soybean phosphatidylcholine, which is rich in linoleic acid, was oxidized with singlet oxygen through photosensitization with methylene blue. This compound facilitates the oxidation of phospholipids relative to the reaction with free unsaturated fatty acids. A response surface methodology was used to control oxidation, with methylene blue concentration and the amount of available air as independent variables. The conjugated diene-to triene ratio was then monitored. Hydroperoxide yield dependent principally on the amount of air, whereas photosensitizer concentration strongly influenced the size and zeta potential of vesicles formed by the sonication of oxidized phospholipids in water. Methylene blue plays an important role in the surface charge expression and ion permeability of these vesicles.