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Basement membrane and cell integrity of self-tissues in maintaining Drosophila immunological tolerance.

Kim MJ, Choe KM - PLoS Genet. (2014)

Bottom Line: The mechanism underlying immune system recognition of different types of pathogens has been extensively studied over the past few decades; however, the mechanism by which healthy self-tissue evades an attack by its own immune system is less well-understood.Here, we established an autoimmune model of melanotic mass formation in Drosophila by genetically disrupting the basement membrane.Moreover, we found that cell integrity, as determined by cell-cell interaction and apicobasal polarity, functions as a second discrete checkpoint.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Yonsei University, Seodaemun-gu, Seoul, South Korea.

ABSTRACT
The mechanism underlying immune system recognition of different types of pathogens has been extensively studied over the past few decades; however, the mechanism by which healthy self-tissue evades an attack by its own immune system is less well-understood. Here, we established an autoimmune model of melanotic mass formation in Drosophila by genetically disrupting the basement membrane. We found that the basement membrane endows otherwise susceptible target tissues with self-tolerance that prevents autoimmunity, and further demonstrated that laminin is a key component for both structural maintenance and the self-tolerance checkpoint function of the basement membrane. Moreover, we found that cell integrity, as determined by cell-cell interaction and apicobasal polarity, functions as a second discrete checkpoint. Target tissues became vulnerable to blood cell encapsulation and subsequent melanization only after loss of both the basement membrane and cell integrity.

No MeSH data available.


Related in: MedlinePlus

Loss of cell integrity is required in addition to BM disruption for melanotic mass formation.(A, B) Mechanical disruption (arrowhead) of the salivary-gland BM of AB1>mys-i, GFP larvae with GFP expression (green) in the salivary gland. (C) Melanotic mass formation in pinch-wounded larvae of AB1>GFP control (n = 1/57) and AB1>mys-i, GFP (n = 17/84) larvae. (D) Melanized salivary glands of wounded AB1>mys-i larvae were positive for L1 (red). Nuclei were stained with DAPI (blue). (E–J) Confocal images of the larval salivary glands of the indicated genotypes after staining lamellocytes with anti-L1 antibodies (red) and nuclei with DAPI (blue). Scale bar: 200 µm (A, B, E–G) and 100 µm (D, H–J).
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pgen-1004683-g005: Loss of cell integrity is required in addition to BM disruption for melanotic mass formation.(A, B) Mechanical disruption (arrowhead) of the salivary-gland BM of AB1>mys-i, GFP larvae with GFP expression (green) in the salivary gland. (C) Melanotic mass formation in pinch-wounded larvae of AB1>GFP control (n = 1/57) and AB1>mys-i, GFP (n = 17/84) larvae. (D) Melanized salivary glands of wounded AB1>mys-i larvae were positive for L1 (red). Nuclei were stained with DAPI (blue). (E–J) Confocal images of the larval salivary glands of the indicated genotypes after staining lamellocytes with anti-L1 antibodies (red) and nuclei with DAPI (blue). Scale bar: 200 µm (A, B, E–G) and 100 µm (D, H–J).

Mentions: To examine the possible role of cell integrity as another checkpoint, we used melanotic mass-free AB1>mys-i larvae. Integrin knockdown with the salivary gland driver AB1-GAL4[42] did not disrupt the BM but resulted in detachment of the BM from the salivary gland tissue (Figure 4K). The cells lost both cell polarity and adhesion properties, and as a result, the BM appeared as a sack containing sticky balls (Figure 4I–K). As expected, hemocytes were not detected on the surface of the salivary gland in these larvae (Figure 4L). To explore this phenotype in more depth, we first mechanically sheared the salivary gland BM by pinching the AB1>mys-i, GFP larva at its anterior side with forceps (Figure 5A and 5B). After two days, these larvae developed black masses in the salivary gland at a rate that was 12-fold higher than that of the pinched control larvae (Figure 5C). Melanized salivary glands dissected from the wounded larvae were positive for L1 (Figure 5D). Second, we enzymatically disrupted the salivary gland BM of ptc>mys-i larvae by overexpressing Mmp2, as a means to more specifically manipulate the larvae. These larvae developed black masses, and the salivary glands dissected from the larvae were positive for L1 (Figure 5E–G). In these experiments, ptc-GAL4 and mys-i27735 were used instead of the previously used AB1-GAL4 and mys-i33642 because the latter combination with UAS-Mmp2 caused severe growth retardation of salivary glands. The reproducibility of the knockdown phenotypes was confirmed using mys-i27735 (Figure S5). Third, we tried to wear out the BM by reducing levels of collagen IV in mys-i larvae. Black masses formed only in the fat bodies of FB>vkg-i, mys-i larvae (Figure 2C): but due to the additional AB1-GAL4 driver, a few of FB+AB1>vkg-i, mys-i larvae developed black masses also in the salivary glands, and again, the salivary glands of those larvae were positive for L1 (Figure 5J). Neither mys-i nor vkg-i alone formed black masses in the salivary gland with the same FB+AB1 GAL4 drivers (Figure 5H and 5I). Taken together, our data demonstrate that cell integrity is an additional and discrete checkpoint for tolerance to self-tissues. We sought to define cell integrity in this system by knocking down genes known to be involved in cell-cell adhesion and cell polarity. Knockdown of either scrib, dlg, cora, FasIII, shg, or arm together with Mmp2 overexpression, however, did not induce melanotic mass formation, indicating that loss of any of these components at least singly did not affect cells sufficiently for disrupting the cell-integrity checkpoint function.


Basement membrane and cell integrity of self-tissues in maintaining Drosophila immunological tolerance.

Kim MJ, Choe KM - PLoS Genet. (2014)

Loss of cell integrity is required in addition to BM disruption for melanotic mass formation.(A, B) Mechanical disruption (arrowhead) of the salivary-gland BM of AB1>mys-i, GFP larvae with GFP expression (green) in the salivary gland. (C) Melanotic mass formation in pinch-wounded larvae of AB1>GFP control (n = 1/57) and AB1>mys-i, GFP (n = 17/84) larvae. (D) Melanized salivary glands of wounded AB1>mys-i larvae were positive for L1 (red). Nuclei were stained with DAPI (blue). (E–J) Confocal images of the larval salivary glands of the indicated genotypes after staining lamellocytes with anti-L1 antibodies (red) and nuclei with DAPI (blue). Scale bar: 200 µm (A, B, E–G) and 100 µm (D, H–J).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199487&req=5

pgen-1004683-g005: Loss of cell integrity is required in addition to BM disruption for melanotic mass formation.(A, B) Mechanical disruption (arrowhead) of the salivary-gland BM of AB1>mys-i, GFP larvae with GFP expression (green) in the salivary gland. (C) Melanotic mass formation in pinch-wounded larvae of AB1>GFP control (n = 1/57) and AB1>mys-i, GFP (n = 17/84) larvae. (D) Melanized salivary glands of wounded AB1>mys-i larvae were positive for L1 (red). Nuclei were stained with DAPI (blue). (E–J) Confocal images of the larval salivary glands of the indicated genotypes after staining lamellocytes with anti-L1 antibodies (red) and nuclei with DAPI (blue). Scale bar: 200 µm (A, B, E–G) and 100 µm (D, H–J).
Mentions: To examine the possible role of cell integrity as another checkpoint, we used melanotic mass-free AB1>mys-i larvae. Integrin knockdown with the salivary gland driver AB1-GAL4[42] did not disrupt the BM but resulted in detachment of the BM from the salivary gland tissue (Figure 4K). The cells lost both cell polarity and adhesion properties, and as a result, the BM appeared as a sack containing sticky balls (Figure 4I–K). As expected, hemocytes were not detected on the surface of the salivary gland in these larvae (Figure 4L). To explore this phenotype in more depth, we first mechanically sheared the salivary gland BM by pinching the AB1>mys-i, GFP larva at its anterior side with forceps (Figure 5A and 5B). After two days, these larvae developed black masses in the salivary gland at a rate that was 12-fold higher than that of the pinched control larvae (Figure 5C). Melanized salivary glands dissected from the wounded larvae were positive for L1 (Figure 5D). Second, we enzymatically disrupted the salivary gland BM of ptc>mys-i larvae by overexpressing Mmp2, as a means to more specifically manipulate the larvae. These larvae developed black masses, and the salivary glands dissected from the larvae were positive for L1 (Figure 5E–G). In these experiments, ptc-GAL4 and mys-i27735 were used instead of the previously used AB1-GAL4 and mys-i33642 because the latter combination with UAS-Mmp2 caused severe growth retardation of salivary glands. The reproducibility of the knockdown phenotypes was confirmed using mys-i27735 (Figure S5). Third, we tried to wear out the BM by reducing levels of collagen IV in mys-i larvae. Black masses formed only in the fat bodies of FB>vkg-i, mys-i larvae (Figure 2C): but due to the additional AB1-GAL4 driver, a few of FB+AB1>vkg-i, mys-i larvae developed black masses also in the salivary glands, and again, the salivary glands of those larvae were positive for L1 (Figure 5J). Neither mys-i nor vkg-i alone formed black masses in the salivary gland with the same FB+AB1 GAL4 drivers (Figure 5H and 5I). Taken together, our data demonstrate that cell integrity is an additional and discrete checkpoint for tolerance to self-tissues. We sought to define cell integrity in this system by knocking down genes known to be involved in cell-cell adhesion and cell polarity. Knockdown of either scrib, dlg, cora, FasIII, shg, or arm together with Mmp2 overexpression, however, did not induce melanotic mass formation, indicating that loss of any of these components at least singly did not affect cells sufficiently for disrupting the cell-integrity checkpoint function.

Bottom Line: The mechanism underlying immune system recognition of different types of pathogens has been extensively studied over the past few decades; however, the mechanism by which healthy self-tissue evades an attack by its own immune system is less well-understood.Here, we established an autoimmune model of melanotic mass formation in Drosophila by genetically disrupting the basement membrane.Moreover, we found that cell integrity, as determined by cell-cell interaction and apicobasal polarity, functions as a second discrete checkpoint.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems Biology, Yonsei University, Seodaemun-gu, Seoul, South Korea.

ABSTRACT
The mechanism underlying immune system recognition of different types of pathogens has been extensively studied over the past few decades; however, the mechanism by which healthy self-tissue evades an attack by its own immune system is less well-understood. Here, we established an autoimmune model of melanotic mass formation in Drosophila by genetically disrupting the basement membrane. We found that the basement membrane endows otherwise susceptible target tissues with self-tolerance that prevents autoimmunity, and further demonstrated that laminin is a key component for both structural maintenance and the self-tolerance checkpoint function of the basement membrane. Moreover, we found that cell integrity, as determined by cell-cell interaction and apicobasal polarity, functions as a second discrete checkpoint. Target tissues became vulnerable to blood cell encapsulation and subsequent melanization only after loss of both the basement membrane and cell integrity.

No MeSH data available.


Related in: MedlinePlus