Roles for cathepsins S, L, and B in insulitis and diabetes in the NOD mouse.

We developed a panel of non-obese diabetic (NOD) mice deficient in major lysosomal cysteine proteases (cathepsins S, L and B) to identify protease enzymes essential for autoimmune diabetes. Null alleles for cathepsins (Cts) S, L or B were introgressed onto the NOD genetic background with 19 Idd markers at homozygosity. Diabetes onset was determined among females aged up to 6 months. We evaluated insulitis and sialadenitis in tissues using histology and computer assisted morphology. NOD mice deficient in Ctss or Ctsb were partially protected from diabetes with incidence at 33% and 28%, respectively, versus wild-type NOD (69%; p < 0.00001). NODs lacking cathepsin L (Ctsl-/-) are completely protected from IDDM, as originally shown by others. Ctsl, Ctss, or Ctsb heterozygous mice were able to develop IDDM, although incidence levels were significantly lower for Ctsb+/- (50%) and Ctsl+/- (55%) as compared to NODs (69%; p < 0.03). Ctsl-/- mice contain functional, diabetogenic T cells and an enriched Foxp3+ regulatory T cell population, and diabetes resistance was due to the presence of an expanded population of regulatory T cells. These data provide additional information about the potency of the diabetogenic T cell population in Ctsl-/- mice which were comparable in potency to wild-type NOD mice. These data illustrate the critical contribution of each of these proteases in determining IDDM in the NOD mouse and provide a useful set of models for further studies.

Coordination of early protective immunity to viral infection by regulatory T cells.

Suppression of immune responses by regulatory T cells (Tregs) is thought to limit late stages of pathogen-specific immunity as a means of minimizing associated tissue damage. We examined a role for Tregs during mucosal herpes simplex virus infection in mice, and observed an accelerated fatal infection with increased viral loads in the mucosa and central nervous system after ablation of Tregs. Although augmented interferon production was detected in the draining lymph nodes (dLNs) in Treg-deprived mice, it was profoundly reduced at the infection site. This was associated with a delay in the arrival of natural killer cells, dendritic cells, and T cells to the site of infection and a sharp increase in proinflammatory chemokine levels in the dLNs. Our results suggest that Tregs facilitate early protective responses to local viral infection by allowing a timely entry of immune cells into infected tissue.

Proteolytic processing of dynamin by cytoplasmic cathepsin L is a mechanism for proteinuric kidney disease.

Kidney podocytes and their foot processes maintain the ultrafiltration barrier and prevent urinary protein loss (proteinuria). Here we show that the GTPase dynamin is essential for podocyte function. During proteinuric kidney disease, induction of cytoplasmic cathepsin L leads to cleavage of dynamin at an evolutionary conserved site, resulting in reorganization of the podocyte actin cytoskeleton and proteinuria. Dynamin mutants that lack the cathepsin L site, or render the cathepsin L site inaccessible through dynamin self-assembly, are resistant to cathepsin L cleavage. When delivered into mice, these mutants restored podocyte function and resolve proteinuria. Our study identifies dynamin as a critical regulator of renal permselectivity that is specifically targeted by proteolysis under pathological conditions.

Cathepsin L stabilizes the histone modification landscape on the Y chromosome and pericentromeric heterochromatin.

Posttranslational histone modifications and histone variants form a unique epigenetic landscape on mammalian chromosomes where the principal epigenetic heterochromatin markers, trimethylated histone H3(K9) and the histone H2A.Z, are inversely localized in relation to each other. Trimethylated H3(K9) marks pericentromeric constitutive heterochromatin and the male Y chromosome, while H2A.Z is dramatically reduced at these chromosomal locations. Inactivation of a lysosomal and nuclear protease, cathepsin L, causes a global redistribution of epigenetic markers. In cathepsin L knockout cells, the levels of trimethylated H3(K9) decrease dramatically, concomitant with its relocation away from heterochromatin, and H2A.Z becomes enriched at pericentromeric heterochromatin and the Y chromosome. This change is also associated with global relocation of heterochromatin protein HP1 and histone H3 methyltransferase Suv39h1 away from constitutive heterochromatin; however, it does not affect DNA methylation or chromosome segregation, phenotypes commonly associated with impaired histone H3(K9) methylation. Therefore, the key constitutive heterochromatin determinants can dynamically redistribute depending on physiological context but still maintain the essential function(s) of chromosomes. Thus, our data show that cathepsin L stabilizes epigenetic heterochromatin markers on pericentromeric heterochromatin and the Y chromosome through a novel mechanism that does not involve DNA methylation or affect heterochromatin structure and operates on both somatic and sex chromosomes.

The lysosomal cysteine proteases in MHC class II antigen presentation.

The endosomal pathway of antigen presentation leads to the display of peptides on major histocompatibility complex (MHC) class II molecules at the cell surface of antigen-presenting cells (APCs). The pathway involves two major steps, invariant chain degradation and antigen processing, which take place in the late endosomes/lysosomes. So far, of the known lysosomal proteases, only cathepsin L and cathepsin S have been shown to have a non-redundant role in endosomal presentation in vivo. Besides being engaged in the degradation of invariant chain, these enzymes also mediate the processing of antigens in distinct cell types. Surprisingly, these enzymes are active in different types of APCs, and this defined expression pattern seems to be enforced by regulatory mechanisms acting on multiple levels.