Bacterial CpG-DNA accelerates Alport glomerulosclerosis by inducing an M1 macrophage phenotype and tumor necrosis factor-α-mediated podocyte loss.

Loss of function mutations in the α3 or α4 chain of type IV collagen cause Alport nephropathy, characterized by progressive glomerulosclerosis. While studying the mechanisms that determine disease progression, we found that the evolution of kidney disease in Col4a3-deficient mice was associated with an influx of immune cell subsets including nonactivated macrophages. This suggested that intrarenal inflammation might accelerate Alport nephropathy. A possible mechanism might be the well-known enhancement of immune recognition by bacterial products. We found that exposure to bacterial endotoxin from 4 to 6 weeks of age did not affect disease progression, whereas an equipotent dose of cytosine-guanine (CpG)-DNA, a synthetic mimic of bacterial DNA, accelerated all aspects of Alport nephropathy and reduced the overall lifespan of Col4a3-deficient mice. This effect was associated with a significant increase of renal CD11b+/Ly6C(hi) macrophages, intrarenal production of inducible nitric oxide synthase, tumor necrosis factor (TNF)-α, interleukin-12, and CXCL10, and loss of podocytes. TNF-α was essential for acceleration of Alport nephropathy, as etanercept (a soluble TNF-α receptor) entirely abrogated the CpG-DNA effect. Thus, systemic exposure to CpG-DNA induces classically activated (M1) macrophages that enhance intrarenal inflammation and disease progression. Hence, factors that modulate the phenotype of renal macrophages can affect the progression of Alport nephropathy and, potentially, other types of chronic kidney diseases.

CpG-DNA upregulates the major acute-phase proteins SAA and SAP.

The acute-phase response is an immediate reaction of the host against invading microorganisms. We show here that oligodeoxynucleotides (ODNs) containing a CpG motif rapidly induce the major murine acute-phase proteins in vivo, i.e. serum amyloid A (SAA) and serum amyloid P (SAP). Serum levels of these proteins are elevated within 12 h and peak at 24 h after the injection of CpG-ODN or endotoxin. Liver cells produce the proteins with the same kinetics. Injection of interleukin 6 (IL-6), IL-1beta and tumour necrosis factor alpha (TNF-alpha) induces SAA and SAP in vivo, but the CpG-ODN-mediated induction does not depend on the presence of the TNF receptor p55, as the acute-phase response in TNF receptor p55-deficient mice does not differ from that of wild-type mice. Aside from CpG-ODN, bacterial genomic DNA also induces the acute-phase response in LPS-resistant C3H/Hej mice. The induction of the major acute-phase proteins SAA and SAP is blocked by the simultaneous injection of CpG-ODN together with D-galactosamine (D-GalN). As D-GalN sensitizes the host for the toxic effects of TNF-alpha, a possible mechanism could be the prevention of synthesis of the major acute-phase proteins SAA and SAP.

CpG motifs in bacterial DNA cause inflammation in the lower respiratory tract.

Since unmethylated CpG motifs are more frequent in DNA from bacteria than vertebrates, and the unmethylated CpG motif has recently been reported to have stimulatory effects on lymphocytes, we speculated that bacterial DNA may induce inflammation in the lower respiratory tract through its content of unmethylated CpG motifs. To determine the role of bacterial DNA in lower airway inflammation, we intratracheally instilled prokaryotic and eukaryotic DNA in C3H/HeBFEJ mice and performed whole lung lavage 4 h after the exposure. Heat denatured, single stranded Escherichia coli genomic DNA (0.06 ng endotoxin/microg DNA) was compared to heat denatured, single stranded calf thymus DNA (0.007 endotoxin/microg DNA). 10 microg of bacterial DNA, in comparison to 10 microg of calf thymus DNA, resulted in a fourfold increase in the concentration of cells (P = 0.0002), a fivefold increase in the concentration of neutrophils (P = 0.0002), a 50-fold increase in the concentration of TNF-alpha (P = 0.001), and a fourfold increase in the concentration of both IL-6 (P = 0.0003) and macrophage inflammatory protein-2 (P = 0.0001) in the lavage fluid. Importantly, instillation of 0.60 ng of E. coli LPS resulted in a negligible inflammatory response. To test whether the stimulatory effects of bacterial DNA are due to its unmethylated CpG dinucleotides, we methylated the bacterial DNA and also prepared 20 base pair oligonucleotides with and without CpG motifs. In comparison to instillation of untreated bacterial DNA, methylation of the bacterial DNA resulted in a significant reduction in the concentration of cells and cytokines in the lower respiratory tract. Moreover, oligonucleotides containing embedded unmethylated CpG motifs resulted in inflammation in the lower respiratory tract that was indistinguishable from that observed with untreated bacterial DNA. In contrast, oligonucleotides without the embedded CpG motifs or with embedded but methylated CpG motifs resulted in significantly less inflammation in the lower respiratory tract. The possible relevance of these data to human disease was shown by extracting and analyzing DNA in sputum from patients with cystic fibrosis (CF). Approximately 0.1 to 1% of this sputum DNA was bacterial. Intratracheal instillation of highly purified CF sputum DNA caused acute inflammation similar to that induced by bacterial DNA. These findings suggest that bacterial DNA, and unmethylated CpG motifs in particular, may play an important pathogenic role in inflammatory lung disease.

[Effect of mammalian and Escherichia coli DNA on rat somatic cell chromosomes]. / Vliianie DNK mlekopitaiushchikh i Escherichia coli na khromosomy somaticheskikh kletok krys

High-polymer DNA isolated from mammalian lymphoid tissues (rat spleen, calf thymus) and from Escherichia coli increases the frequency of quantitative lesions in bone marrow cells of normal Wistar rats. The highest percentage of aberrant metaphases was revealed 24 hours after the injection of mammalian DNA, the frequency of aberrations being 9 times higher than the control values after the injection of heterologous DNA and 6 times higher-after the injection of homologous DNA. The effect observed was not a prolonged one, and 72 hours following the DNA injection the numbers of aberrant cells decreased to the control level. The maximal frequency of aberrations in bone marrow cells of rats treated with bacterial DNA was found 72 hours after the injection, when a 4-fold increase above the spontaneous aberration level was observed. Definite differences in the character of structural changes of chromosomes induced by DNA of different origin were revealed. Mammalian DNA injected produced the chromatid-type aberrations only. The injection of bacterial DNA led to the formation of both chromatid and chromosome aberrations. Possible mechanisms of the increase of chromosome aberration frequency in rat somatic cells under the action of high-polymer DNA of different origin are discussed.