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

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

Co-infection does not prevent Th1 and Th2 cell polarization.

<p>Splenocytes from mice which were infected as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003341#pntd-0003341-g001" target="_blank">Figure 1A</a> were re-stimulated ex-vivo with anti-CD3 in the presence of anti-CD28: IFNγ and IL-13 induction in <b>A</b>) CD3⁺CD4⁺ T cells and <b>B</b>) CD3⁻CD4⁻ cells was measured 6 hours post-stimulation by intracellular FACS and is represented as a proportion and/or absolute numbers. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Data is representative of 4–6 mice per group with experiments performed twice for each time point. (NS = Non-significant, ^*P<0.05 and ^**P<0.005).</p

Prior Infection with Nb alters the control of STm and production of anti-STm IgG.

<p>WT mice were infected with 500 L3 Nb larvae and at day 16 mice were challenged with 5×10⁵ STm alongside naïve control mice. Splenic bacterial numbers were assessed at days 5 and 25 post-STm infection. Serum anti-STm IgM, IgG, IgG2a and IgG2b antibody titres were assessed by ELISA against a total outer membrane preparation of STm. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Groups contained 4–6 mice. (^*P<0.05 and ^**P<0.005).</p

Co-infection alters the frequency of T-zone localised FoxP3 cells.

<p>Spleen sections were generated for immunohistology at day 5 post-infection from mice which were infected as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003341#pntd-0003341-g001" target="_blank">Figure 1A</a>. Sections were double-stained for FoxP3 with CD3. These sections were then used to quantify FoxP3+CD3+ T cells in the T-zone per mm². Representative images show double-staining with FoxP3 (blue) and IgD (brown) with images acquired using a Leica microscope DM6000 using a 20× objective. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Data is representative of 4–6 mice per group with experiments performed twice for each time point. T = T zone; F = B cell follicle (^*P<0.05 and ^**P<0.005).</p