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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 17, 2001 as doi:10.1096/fj.01-0306fje. |
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Immunobiology Section, Laboratory of Parasitic Diseases and the
* Laboratory of Immunopathology; National Institutes of Allergy and Infectious Diseases and the
Cancer Genetics Branch, National Human Genome Research Institute at the National Institutes of Health, Bethesda, Maryland 20892, USA; and the
Biomedical Research Institute, Rockville, Maryland 20852, USA
2Correspondence: National Institutes of Health, 9000 Rockville Pike, Bldg. 4, Room 126, Bethesda, MD 20892-0425, USA. E-mail: twynn{at}niaid.nih.gov
SPECIFIC AIMS
In this study, we addressed the hypothesis that severe infection-induced liver disease can develop via two distinct but equally deleterious genetic programs depending on the dominant type 1 or type 2 immunological phenotype. By using murine cDNA microarrays, cytokine-deficient mice, and confirmatory biological assays, we identified gene expression profiles that associate with lethal type 1 and type 2 cytokine polarized immune responses and uncovered the contributions of previously unappreciated disease mechanisms to pathogenesis during infection with the parasitic trematode Schistosoma mansoni.
PRINCIPAL FINDINGS
1. Increased expression of genes involved in hepatic matrix remodeling, tissue repair, and collagen deposition are associated with enhanced hepatic fibrosis and increased mortality in infected type 2 polarized mice
Initiation of egg laying (5 wk postinfection) in schistosome-infected interleukin 10 (IL-10)/IL-12 (type 2 polarized) and IL-10/IL-4 (type 1 polarized) -deficient mice induced dramatic differences in transcription of genes involved in the processes associated with collagen deposition, wound repair, and matrix remodeling. The type 2 polarized mice had the lowest expression of these genes before the onset of egg laying and the highest expression 6.5 and 8 wk postinfection (Fig. 1
A–E). In support of these observations, liver hydroxyproline, a quantitative measure of fibrosis, was elevated 8 wk postinfection in the type 2 polarized mice and suppressed in the type 1 polarized animals (Fig. 1F
) when compared with (wild-type) WT. Collagen staining of liver granulomas also confirmed these findings (Fig. 1G
). Thus, these results clearly illustrate that type 2 responses promote and type 1 responses inhibit gene transcription pathways associated with liver collagen synthesis and matrix remodeling. Furthermore, they provide a mechanistic explanation for the increased mortality rates reported earlier for the type 2 polarized exacerbated liver fibrosis and portal hypertension.
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2. cDNA microarrays predict differential recruitment of macrophages, neutrophils, and eosinophils into the livers of type 1 and type 2 polarized mice
Our cDNA microarray data predicted there would be differences in the recruitment of granulocytes into the livers of the immune polarized and WT control mice. In support of these predictions, there were significantly more liver eosinophils observed in both type 2 polarized and WT mice than in the type 1 polarized animals as determined by fluorescein-activated cell sorter (FACS). In support of the cDNA microarray predictions, there were more neutrophils recruited to livers of mice with either double cytokine-deficient genotype. Finally, we observed increased expression of macrophage-associated transcripts in the livers of type 1 polarized mice during infection. FACS analysis of liver leukocytes 8 wk postinfection confirmed there were many more liver macrophages present in the type 1 polarized mice than the other two groups. The results from these findings suggest that the aberrant recruitment of granulocytes and expression of their associated transcripts likely contribute to the lethal immunopathology observed in the type 1 and type 2 polarized mice.
3. Increased recruitment of activated neutrophils and macrophages into livers of type 1 polarized mice is associated with enhanced programmed cell death and mortality rates
We found several genes involved in the induction of apoptosis rapidly up-regulated in the livers of infected type 1 polarized mice. Conversely, genes involved in the inhibition of apoptosis were up-regulated to a greater extent in the livers of IL-10/IL-12-deficient and WT mice. From these expression data, we predicted that the mechanisms controlling programmed cell death, possibly influenced by the abnormal recruitment of granulocytes, were up-regulated in the infected type 1 polarized animals. Although TUNEL-positive cells were found in nearly every granuloma examined independent of mouse genotype, the type 1 polarized mice consistently displayed significantly more, confirming the cDNA microarray predictions. Therefore, in addition to the severe and rapid cachexia, elevated liver transaminase activity, and high proinflammatory cytokine production previously reported in the infected type 1 polarized mice, we found aberrant regulation of the apoptotic machinery. The combination of these factors likely explains the rapid morbidity and mortality observed for the type 1 polarized mice after infection with S. mansoni.
CONCLUSIONS
The results of this study demonstrate two major points. First, cDNA microarrays can be used to successfully characterize differential patterns of gene expression in distinct disease states. Second, and of equal importance, these data are able to uncover contributions of previously unappreciated disease mechanisms to pathogenesis. Together these findings provide an increased richness and depth to our understanding of the mechanisms involved in schistosome-induced liver pathology (Fig. 2
).
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This study also provides a detailed view of the genetic events that characterize the development of severe liver pathology. Because unique patterns of gene expression were observed in our type 1 and type 2 polarized mice, these findings may be more broadly applicable to other diseases where expression of a polarized immune response is responsible for pathology. Hepatic fibrosis is the key pathological change in schistosomiasis, and the findings presented here demonstrate that type 2 cytokines are critical for this response. These cDNA microarray findings also demonstrate that uncontrolled granulocyte recruitment and transcriptional activation can contribute to pathological inflammatory lesions. Several genes expressed preferentially during polarized type 1 and type 2 cytokine responses were identified in this study, and our future experiments will focus on assessing their function in schistosomiasis. Given their unique expression profiles, the proteins en-coded by many of these genes may also serve as useful ‘morbidity markers’ for severe schistosomiasis and possibly other chronic inflammatory diseases. DNA microarrays hold the promise of mapping disease pathways in fine detail; the results shown here nicely illustrate the potential of genomic approaches for generating comprehensive views of the mechanisms regulating infectious disease pathogenesis.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0306fje; to cite this article, use FASEB J. (September 17, 2001) 10.1096/fj.01-0306fje 
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