The potential roles of reactive nitrogen species in host defence against pathogens and in human health and autoimmune inflammatory diseases

Abstract

The human immune system defends the body against infectious pathogens such as bacteria and viruses. In humans, oral bacteria reduce dietary nitrate (NO3ˉ) to nitrite (NO2ˉ). NO2ˉ is swallowed into the acidic stomach environment, and is converted to bactericidal reactive nitrogen species (RNS). But the effects of RNS on viruses are poorly understood. In this thesis, the first research study tested the antimicrobial effects of varying pH, in the absence/presence of NO2ˉ, on E. coli growth and on the replication of the lambda phage virus. The antimicrobial activities were determined by viability/plaque assays. Buffers themselves, in the absence of NO2ˉ, had statistically significant inhibitory effects on E. coli growth when the E. coli was exposed to the KH phthalate-HCl buffer at pH 3.0, glycine-HCl buffer at pH 3.0, citrate buffer at pH’s 3.0, 5.0, and 6.0, and phosphate buffer at pH’s 6.0 and 7.4. These systems also had statistically significant inhibitory effects on lambda phage replication at the same pH values except phosphate buffer at pH 7.4. RNS from acidified NO2ˉ had antibacterial effects towards E. coli, compared with the effect of acid pH alone. NO2ˉ in citrate buffer at pH 3.0 had no antiviral effect on lambda phage. Stomach NO2ˉ acidification is bactericidal, but may not inhibit lambda phage replication.

White blood cells (WBCs) aid the immune system’s defences against infection, in part by generating nitric oxide (˙NO) which is oxidised to NO3ˉ and NO2ˉ. The second study measured the concentrations of NO3ˉ and NO2ˉ in lysates of peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) from healthy volunteers. Concentrations of NO3ˉ and NO2ˉ were measured by ozone-based chemiluminescence. The median concentrations of NO3ˉ and NO2ˉ, when expressed per 106 cells, or per ml of packed cells were significantly higher in PBMCs and PMNs compared to erythrocytes. These high concentrations may reflect a relatively high cellular ˙NO production, and/or a cellular substrate pool which may be convertible to ˙NO.

Increased inflammatory generation of superoxide (O2˙⁻) and ˙NO is associated with increased levels of 3-nitrotyrosine (3-NT) residues within proteins. But the presence and distribution of 3-NT within WBC proteins in human autoimmune inflammatory diseases is poorly characterised. The third study detected 3-NT in PBMC lysates from healthy volunteers and patients with rheumatoid arthritis, systemic lupus erythematosus, primary Sjogren’s syndrome and scleroderma. The total protein 3-NT levels were quantified by a high-sensitivity electrochemiluminescence-based immunosorbent assay (ECLISA), whilst the distribution of 3-NT, as a constituent of specific cellular proteins was detected by western blotting. In PBMC lysates, the dominant nitrated protein was β-actin. The median values of the normalised nitrated β-actin band intensities were significantly higher in all autoimmune inflammatory groups compared to the healthy control group. The increased nitration of β-actin may play a role in the pathogenesis of inflammatory diseases.

The present research confirmed the potential effect of RNS, from acidified NO2ˉ, against bacterial infection, but found no evidence for an antiviral effect. The identified reservoir of NO3ˉ/NO2ˉ in PBMCs/PMNs may provide a substrate source for cellular ˙NO formation. The detected formation of 3-NT within the cytoskeletal β-actin of PBMCs may have implications for immune cell function.