Mucopolysaccharidosis IIIB, a lysosomal storage disease, triggers a pathogenic CNS autoimmune response

<p>Abstract</p> <p>Background</p> <p>Recently, using a mouse model of mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease with severe neurological deterioration, we showed that MPS IIIB neuropathology is accompanied by a robust neuroinflammatory response of unknown consequence. This study was to assess whether MPS IIIB lymphocytes are pathogenic.</p> <p>Methods</p> <p>Lymphocytes from MPS IIIB mice were adoptively transferred to naïve wild-type mice. The recipient animals were then evaluated for signs of disease and inflammation in the central nervous system.</p> <p>Results</p> <p>Our results show for the first time, that lymphocytes isolated from MPS IIIB mice caused a mild paralytic disease when they were injected systemically into naïve wild-type mice. This disease is characterized by mild tail and lower trunk weakness with delayed weight gain. The MPS IIIB lymphocytes also trigger neuroinflammation within the CNS of recipient mice characterized by an increase in transcripts of IL2, IL4, IL5, IL17, TNFα, IFNα and Ifi30, and intraparenchymal lymphocyte infiltration.</p> <p>Conclusions</p> <p>Our data suggest that an autoimmune response directed at CNS components contributes to MPS IIIB neuropathology independent of lysosomal storage pathology. Adoptive transfer of purified T-cells will be needed in future studies to identify specific effector T-cells in MPS IIIB neuroimmune pathogenesis.</p

Human Alpha Defensin 5 Expression in the Human Kidney and Urinary Tract

The mechanisms that maintain sterility in the urinary tract are incompletely understood. Recent studies have implicated the importance of antimicrobial peptides (AMP) in protecting the urinary tract from infection. Here, we characterize the expression and relevance of the AMP human alpha-defensin 5 (HD5) in the human kidney and urinary tract in normal and infected subjects.Using RNA isolated from human kidney, ureter, and bladder tissue, we performed quantitative real-time PCR to show that DEFA5, the gene encoding HD5, is constitutively expressed throughout the urinary tract. With pyelonephritis, DEFA5 expression significantly increased in the kidney. Using immunoblot analysis, HD5 production also increased with pyelonephritis. Immunostaining localized HD5 to the urothelium of the bladder and ureter. In the kidney, HD5 was primarily produced in the distal nephron and collecting tubules. Using immunoblot and ELISA assays, HD5 was not routinely detected in non-infected human urine samples while mean urinary HD5 production increased with E.coli urinary tract infection.DEFA5 is expressed throughout the urinary tract in non-infected subjects. Specifically, HD5 is expressed throughout the urothelium of the lower urinary tract and in the collecting tubules of the kidney. With infection, HD5 expression increases in the kidney and levels become detectable in the urine. To our knowledge, our findings represent the first to quantitate HD5 expression and production in the human kidney. Moreover, this is the first report to detect the presence of HD5 in infected urine samples. Our results suggest that HD5 may have an important role in maintaining urinary tract sterility

Innate and Adaptive Immune Activation in the Brain of MPS IIIB Mouse Model

Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disease with severe neurological manifestations due to a-N-acetylglucosaminidase (NaGlu) deficiency. The mechanism of neuropathology in MPS IIIB is unclear. This study investigates the role of immune responses in neurological disease of MPS IIIB in mice. By means of gene expression microarrays and realtime quantitative reverse transcriptase-polymerase chain reaction, we demonstrated significant up-regulation of numerous immune-related genes in MPS IIIB mouse brain involving a broad range of immune cells and molecules, including T cells, B cells, microglia/ macrophages, complement, major histocompatibility complex class I, immunoglobulin, Toll-like receptors, and molecules essential for antigen presentation. The significantly enlarged spleen and lymph nodes in MPS IIIB mice were due to an increase in splenocytes/lymphocytes, and functional assays indicated that the T cells were activated. An autoimmune component to the disease was further suggested by the presence of putative autoantigen or autoantigens in brain extracts that reacted specifically with serum IgG from MPS IIIB mice. We also demonstrated for the first time that immunosuppression with prednisolone alone can significantly slow the central nervous system disease progression. Our data indicate that immune responses contribute greatly to the neuropathology of MPS IIIB and should be considered as an adjunct treatment in future therapeutic developments for optimal therapeutic effect

Urinary production of HD5 in infected urine samples.

<p>HD5 levels in sterile urine samples and urine samples infected with uropathogenic <i>E.coli</i>. The square boxes depict urinary proHD5 standardized to urine creatinine detected by ELISA assay using monoclonal antibody 8C8. The open circles depict urinary proHD5 and mature HD5 standardized to urine creatinine by immunoblot analysis. ProHD5 and mature HD5 were not detected in sterile urine samples using polyclonal HD5 antisera.</p

HD5 production in non-infected human kidney and kidney with pyelonephritis.

<p>Immunohistochemistry demonstrates HD5 production in isolated renal tubules (brown/arrows) in non-infected renal cortex (<b>A</b>) and medulla (<b>C</b>). With pyelonephritis, HD5 production increased in the renal tubules of the cortex (<b>B</b>) and medulla (<b>D</b>). The glomeruli (+) show no immunostaining in non-infected kidney samples and with pyelonephritis. Negative controls showed no immunostaining (not shown). Magnification 20×.</p

Tubular HD5 expression in states of sterility and infection.

<p>Human kidney labeled for HD5 (green), nuclei (blue) and nephron specific markers (red). Segment markers consisted of AQP-2 for collecting tubules (CT), THP for the loop of Henle (LOH), and AQP-1 for proximal tubules (PT). <b>A/B</b>: HD5 (green) was produced in the collecting tubules (red apical AQP-2 staining) of non-infected kidney tissue (A) and with pyelonephritis (B). Arrows indicate HD5 is produced in other nephron segments. <b>C/D</b>: HD5 (green) was expressed in the loops of Henle (red THP staining) in non-infected kidney tissue (C) and with pyelonephritis (D). <b>E/F</b>: HD5 (green) shows minimal production in the proximal tubules (red AQP-1 staining) of non-infected kidney tissue (E) and with pyelonephritis (F). Magnification 40×.</p

Expression of <i>DEFA5</i> in human kidney, ureter, and bladder.

<p><i>DEFA5</i> mRNA transcript levels were quantified by real-time PCR in non-infected kidney, ureter, bladder. Shown are the results of three independent samples. In the table below, the mean transcript levels are shown with the SEM. <i>DEFA5</i> expression was significantly greater in the lower urinary tract (<i>p</i> = 0.014).</p

HD5 expression increases with pyelonephritis.

<p>(<b>A</b>) <i>DEFA5</i> mRNA transcript levels were quantified by real-time PCR in non-infected kidney tissue and in kidney tissue with pyelonephritis. Shown are the results for three independent samples. In the table below, the mean transcript levels are shown with the SEM. <i>DEFA5</i> expression was significantly greater with pyelonephritis (<i>p</i> = 0.019). (<b>B</b>) To confirm the increase in message is accompanied by an increase in HD5 protein production, cationic peptides from the same non-infected kidney tissues (NL) and kidney tissue with pyelonephritis (P) were subjected to SDS PAGE followed by Western immunoblot analysis. Each lane contained the equivalent of 800 µg of cationic protein. Silver stained PAGE gels (top panel) confirmed equal protein loading into each lane. Immunoblot analysis for GAPDH and HD5 (middle panel). Serial dilutions of proHD5 (200 ng–70 ng) were subjected to SDS PAGE followed by Western immunoblot analysis (bottom panel).</p

HD5 is expressed throughout the urothelium of the ureter and bladder.

<p>Immunohistochemistry demonstrates HD5 expression (brown/arrows) throughout the urothelium of the human bladder (<b>A</b>) and ureter (<b>C</b>). Immunostaining was most prominent in the luminal surfaces (brown/arrows). Immunostaining was not detected in the smooth muscle layers of the bladder or ureter (+). Negative control bladder (<b>B</b>) and ureter (<b>D</b>) showed no immunostaining. Magnification 20×.</p