Identifying the molecular basis for treatment resistance in a subset of myasthenia gravis patients of African ancestory

Includes abstract.Includes bibliographical references.Myasthenia gravis (MG) is an autoimmune disease in which pathogenic antibodies block, target or destroy the acetylcholine receptors of the muscle endplate resulting in failure of neuromuscular transmission and fatigable weakness. We have previously shown that a subpopulation of South African MG patients of African genetic ancestry develop a severe extraocular muscle (EOM) phenotype whilst receiving standard immunosuppressive drug therapies. This phenotype associates with a functional c.-198C>G SNP (C>G SNP) in the regulatory region of decay accelerating factor (DAF), a complement regulatory protein, which is essential for protection against complementmediated damage. MG patients are treated with prednisone as the first-line immunosuppressant and frequently, an additional steroid-sparing agent, such as azathioprine, cyclosporin A or methotrexate. We hypothesised that MG patients with the C>G SNP when treated with immunosuppressant drugs, may have lower DAF protein levels contributing to increased susceptibility to autologous complement-mediated damage at their EOMs. This study tests this by comparing the effect of prednisone, azathioprine, cyclosporine and methotrexate individually and prednisone in combination with each of these steroid-sparing agents on wild-type and C>G DAF protein (western blotting) and mRNA (quantitative real time PCR) levels and promoter activity (luciferase reporter assays). These experiments were performed in EBV transformed lymphoblastoid cell lines from control individuals with wild-type DAF and MG patients carrying the C>G SNP. As a more representative model for this study the experiments were repeated in mouse skeletal muscle cells because there was no available EOM cell line. In support of the hypothesis of this study, prednisone, cyclosporin A and azathioprine individually was shown to repress C>G DAF protein levels while having either no effect on wild-type DAF or slightly activating it. Importantly, methotrexate was the only drug that was able to increase C>G DAF lymphoblast protein expression and therefore holds promise as a potentially effective treatment for MG patients with the C>G SNP. Moreover, using a calcein assay, clinically relevant doses of prednisone in combination with MG patient sera was shown to significantly increase the susceptibility of C>G DAF lymphoblasts to cell lysis (82% C>G DAF lymphoblasts vs. 9% wild-type DAF lymphoblasts). These results suggest that MG patient sera contain factor(s) that together with prednisone may also be responsible for the susceptibility of the EOMs in these patients to injury. The results show that the levels of DAF protein and mRNA did not always match which suggests that the drugs tested may regulate the DAF protein at a posttranscriptional level. In a mouse skeletal muscle model, Daf (equivalent to human wild-type DAF) protein expression was consistently repressed by prednisone treatment but activated by cyclosporine A, azathioprine and methotrexate. Furthermore, co-treatment of prednisone with either of the steroid-sparing drugs showed that azathioprine, and low doses of cyclosporin A and methotrexate were able to overcome the repressive effect of prednisone-only treatment on Daf expression. These observations indicate that the regulation of Daf may be species and/or cell-type specific. Together the results from this thesis suggest that in EOMs, where DAF is already downregulated, compared to other muscles, our MG patients with the C>G SNP may have an increased susceptibility to complement-mediated damage when treated with prednisone, which further represses C>G DAF expression

Surfactant Protein-D is essential for immunity to Helminth Infection

Author Summary Infections by parasitic worms are very common, and controlling them is a major medical and veterinary challenge. Very few drugs exist to treat them, and the parasites can develop resistance to these. In order to find new ways to control worm infections, understanding how our immune system responds to them is essential. Many important parasitic worm infections move through the host lung. In this study we show that a major secreted protein in the lung, Surfactant Protein D (SP-D), is essential for immunity to a parasitic worm infection. We found that this protein binds to worm larvae in the lung to help the immune system kill them. Infecting mice that do not express SP-D with worms demonstrates SP-D is important in this immune response. These mice are unable to launch an effective anti-worm immune response and have many more worms in their intestine compared to mice that do express SP-D. We also show that if we increase SP-D levels in the lung the mouse has better immunity to worms. Together this shows for the first time that SP-D is very important for immunity to worm infections

Natural and Vaccine-Mediated Immunity to Salmonella Typhimurium is Impaired by the Helminth Nippostrongylus brasiliensis

The impact of exposure to multiple pathogens concurrently or consecutively on immune function is unclear. Here, immune responses induced by combinations of the bacterium Salmonella Typhimurium (STm) and the helminth Nippostrongylus brasiliensis (Nb), which causes a murine hookworm infection and an experimental porin protein vaccine against STm, were examined. Mice infected with both STm and Nb induced similar numbers of Th1 and Th2 lymphocytes compared with singly infected mice, as determined by flow cytometry, although lower levels of secreted Th2, but not Th1 cytokines were detected by ELISA after re-stimulation of splenocytes. Furthermore, the density of FoxP3+ T cells in the T zone of co-infected mice was lower compared to mice that only received Nb, but was greater than those that received STm. This reflected the intermediate levels of IL-10 detected from splenocytes. Co-infection compromised clearance of both pathogens, with worms still detectable in mice weeks after they were cleared in the control group. Despite altered control of bacterial and helminth colonization in co-infected mice, robust extrafollicular Th1 and Th2-reflecting immunoglobulin-switching profiles were detected, with IgG2a, IgG1 and IgE plasma cells all detected in parallel. Whilst extrafollicular antibody responses were maintained in the first weeks after co-infection, the GC response was less than that in mice infected with Nb only. Nb infection resulted in some abrogation of the longer-term development of anti-STm IgG responses. This suggested that prior Nb infection may modulate the induction of protective antibody responses to vaccination. To assess this we immunized mice with porins, which confer protection in an antibody-dependent manner, before challenging with STm. Mice that had resolved a Nb infection prior to immunization induced less anti-porin IgG and had compromised protection against infection. These findings demonstrate that co-infection can radically alter the development of protective immunity during natural infection and in response to immunization

Inactivation of glutathione peroxidase activity contributes to UV-induced squamous cell carcinoma formation

Cutaneous squamous cell carcinomas (CSCC) are a common malignancy of keratinocytes that arise in sites of the skin exposed to excessive UV radiation. In the present study, we show that human SCC cell lines, preneoplastic solar keratoses (SK), and CSCC are associated with perturbations in glutathione peroxidase (GPX) activity and peroxide levels. Specifically, we found that two of three SKs and four of five CSCCs, in vivo, were associated with decreased GPX activity and all SKs and CSCCs were associated with an elevated peroxide burden. Given the association of decreased GPX activity with CSCC, we examined the basis for the GPX deficiency in the CSCCs. Our data indicated that GPX was inactivated by a post-translational mechanism and that GPX could be inactivated by increases in intracellular peroxide levels. We next tested whether the decreased peroxidase activity coupled with an elevated peroxidative burden might contribute to CSCC formation in vivo. This was tested in Gpx1–/– and Gpx2–/– mice exposed to solar-simulated UV radiation. These studies showed that Gpx2 deficiency predisposed mice to UV-induced CSCC formation. These results suggest that inactivation of GPX2 in human skin may be an early event in UV-induced SCC formation

Immunity to <i>N</i>. <i>brasiliensis</i> is impaired in SP-D-/- mice.

<p><i>N</i>. <i>brasiliensis</i> infected wild type and SP-D-/- mice were killed at days 9 and 16 PI. Intestinal worm burdens were quantified <b>(a)</b>. Levels of lung and intestinal tissue IL-13 <b>(b)</b>, IL-33, TSLP and IL-25 <b>(c)</b> were established by ELISA at days 9 and 16 PI. FACS analysis established proportions and total numbers of ILC <b>(d)</b>. Numbers and proportions of alveolar macrophage and mean fluorescence intensity (MFI) of RELM-α detection within this population <b>(e).</b> Relative concentration of RELM-α in the lungs of day 9PI WT and SP-D-/- mice was detected by Western Blot and quantified by densitometry <b>(f).</b> Data are representative of 2–3 individual experiments with N = 4–6 mice per group. *P<0.05, **P<0.01.</p

Selective SP-D binding to L4 <i>Nippostrongylus brasiliensis</i> enhances host protective immunity.

<p>Confocal microscopic images of L3, L4 and Adult stage of <i>N</i>. <i>brasiliensis</i> incubated with 20 μg/ml of rfhSP-D, biotin-conjugated anti-rfhSP-D, followed by streptavidin-cy3 <b>(a)</b>. 250 L4 stage larvae, coated or uncoated with rfhSP-D were intra-nasally administered into naïve mice. Intestinal worm burden <b>(b)</b>, detection by ELISA of IL-33, TSLP and IL-25 in the lung <b>(c)</b>, percentage of lung ILC2 and mean fluorescence intensity (MFI) of markers of alternative macrophage activation were quantified at day 5 PI. <b>(d)</b>. Data are representative of 4 individual experiments. N = 5–8 mice per group. *P<0.05, **P<0.01.</p

Elevation of SP-D in BAL fluid following <i>N</i>. <i>brasiliensis</i> infection is dependent on IL-4/IL-13 and IL-4Rα.

<p>SP-D levels were measured by ELISA in BAL fluid at various time points post <i>N</i>. <i>brasiliensis</i> infection (<b>a</b>). SP-D levels in IL-4/IL-13^-/- (<b>b</b>) and IL-4R<b>α</b>^-/- mice (<b>c</b>) were measured in BAL fluid at day 5 PI and compared to wild type controls. Data are representative of 3 individual experiments. N = 5 mice per group. *P<0.05, **P<0.01.</p

SP-D-mediated protection against <i>N</i>. <i>brasiliensis</i> infection requires carbohydrate recognition domain binding to opsonise L4 for enhanced macrophage killing.

<p>Mice were treated with BSA, rfhSP-D or maltose bound rfhSP-D. Intestinal worm burdens were quantified at day 5 PI, and alveolar cell populations were determined at day 5 PI (<b>a</b>). Untreated <i>N</i>. <i>brasiliensis</i> L4 or <i>N</i>. <i>brasiliensis</i> L4 pre-incubated with 20 μg/ml SP-D were cultured without or with alveolar macrophages (isolated from day 7 <i>N</i>. <i>brasiliensis</i> infected mice) for 48 hrs in serum free media and analysed by microscopy for viability (<b>b</b>). Top row shows bright field, bottom row shows standard deviation of overlay of 20 sequence pictures; white indicates movement. Data are representative of two individual experiments. N = 5 mice per group. *P<0.05, **P<0.01.</p

Intra-nasal administration of SP-D enhances protective immunity to <i>N</i>. <i>brasiliensis</i>.

<p>rfhSP-D treated or untreated mice infected with <i>N</i>. <i>brasiliensis</i> were killed at day 5 PI. (<b>a</b>). Intestinal worm burdens were quantified at day 5 PI. (<b>b</b>). IL-4, IL-13 and IL-33 cytokine levels in lung homogenates were detected by ELISA at day 5 PI. (<b>c</b>). Total numbers, total percentage and percentage of IL-13 positive lung ILC2s <b>(d)</b> were quantified by FACS analysis at D5 PI Black bars: control mice. White bars: SP-D treated mice. Data are representative of 2–3 experiments. N = 5–6 mice per group. *P<0.05, **P<0.01.</p