Search

Abstract Background Ribonuclease 8 is a member of the RNase A family of secretory ribonucleases; orthologs of this gene have been found only in primate genomes. RNase 8 is a divergent paralog of RNase 7, which is lysine-enriched, highly conserved, has prominent antimicrobial activity, and is expressed in both normal and diseased skin; in contrast, the physiologic function of RNase 8 remains uncertain. Here, we examine the genetic diversity of human RNase 8, a subject of significant interest given the existence of functional pseudogenes (coding sequences that are otherwise intact but with mutations in elements crucial for ribonucleolytic activity) in non-human primate genomes. Results RNase 8 expression was detected in adult human lung, spleen and testis tissue by quantitative reverse-transcription PCR. Only two single-nucleotide polymorphisms and four unique alleles were identified within the RNase 8 coding sequence; nucleotide sequence diversity (π = 0.00122 ± 0.00009 per site) was unremarkable for a human nuclear gene. We isolated transcripts encoding RNase 8 via rapid amplification of cDNA ends (RACE) and RT-PCR which included a distal potential translational start site followed by sequence encoding an additional 30 amino acids that are conserved in the genomes of several higher primates. The distal translational start site is functional and promotes RNase 8 synthesis in transfected COS-7 cells. Conclusions These results suggest that RNase 8 may diverge considerably from typical RNase A family ribonucleases and may likewise exhibit unique function. This finding prompts a reconsideration of what we have previously termed functional pseudogenes, as RNase 8 may be responding to constraints that promote significant functional divergence from the canonical structure and enzymatic activity characteristic of the RNase A family.

Abstract Background The innate immune responses of neonates differ dramatically from those of adults. Here we examine the acute inflammatory responses of neonatal and weanling mice infected with pneumonia virus of mice (PVM), a rodent pathogen (family Paramyxoviridae, genus Pneumovirus) that replicates the sequelae of severe respiratory syncytial virus infection. Results We demonstrate that virus replication proceeds indistinguishably in all age groups (inoculated at 1, 2, 3 and 4 weeks of age), although inflammatory responses vary in extent and character. Some of the biochemical mediators detected varied minimally with age at inoculation. Most of the mediators evaluated demonstrated elevated expression over baseline correlating directly with age at the time of virus inoculation. Among the latter group are CCL2, CCL3, and IFN-γ, all cytokines previously associated with PVM-induced inflammatory pathology in mature mice. Likewise, we detect neutrophil recruitment to lung tissue in all age groups, but recruitment is most pronounced among the older (3 - 4 week old) mice. Interestingly, all mice exhibit failure to thrive, lagging in expected weight gain for given age, including the youngest mice that present little overt evidence of inflammation. Conclusions Our findings among the youngest mice may explain in part the phenomenon of atypical or minimally symptomatic respiratory infections in human neonates, which may be explored further with this infection model.

Abstract Background We have shown previously that acute infection with the respiratory pathogen, pneumonia virus of mice (PVM), results in local production of the proinflammatory chemokine, CCL3, and that neutrophil recruitment in response to PVM infection is reduced dramatically in CCL3 -/- mice. Results In this work, we demonstrate that CCL3-mediated neutrophil recruitment is coordinated by interferon-gamma (IFNγ). Neutrophil recruitment in response to PVM infection was diminished five-fold in IFNγ receptor gene-deleted mice, although neutrophils from IFNγR -/- mice expressed transcripts for the CCL3 receptor, CCR1 and responded functionally to CCL3 ex vivo. Similarly, in the absence of PVM infection, CCL3 overexpression alone could not elicit neutrophil recruitment in the absence of IFNγ. Interestingly, although supplemental IFNγ restored neutrophil recruitment and resulted in a sustained weight loss among CCL3-overexpressing IFNγ -/- mice, CCL3-mediated neutrophil recruitment alone did not result in the pulmonary edema or respiratory failure characteristic of severe viral infection, suggesting that CCL3 and IFN-γ together are sufficient to promote neutrophil recruitment but not pathologic activation. Conclusion Our findings reveal a heretofore unrecognized hierarchical interaction between the IFNγ and CCL3, which demonstrate that IFNγ is crucial for CCL3-mediated neutrophil recruitment in vivo.