In and out: Leishmania metastasis by hijacking lymphatic system and migrating immune cells.

The lymphatic system plays a crucial role in mounting immune response against intracellular pathogens, and recent studies have documented its role in facilitating tumor dissemination linked largely with cancer cells. However, in mucocutaneous leishmaniasis (MCL) caused by Leishmania Viannia subgenus showing infectious metastasis and resulting in severe distant secondary lesions, the route of escape of these parasites to secondary sites has not yet been investigated in detail. Our results demonstrated that when infection was associated with inflammation and additionally exacerbated by the presence of dsRNA viral endosymbiont (LRV1), lymphatic vessels could serve as efficient routes for infected cells to egress from the primary site and colonize distant organs. We challenged this hypothesis by using the intracellular Leishmania protozoan parasites Leishmania guyanensis (Lgy) associated with or without a dsRNA viral endosymbiont, exacerbating the infection and responsible for a strong inflammatory response, and favoring metastasis of the infection. We analyzed possible cargo cells and the routes of dissemination through flow cytometry, histological analysis, and in vivo imaging in our metastatic model to show that parasites disseminated not only intracellularly but also as free extracellular parasites using migrating immune cells, lymph nodes (LNs), and lymph vessels, and followed intricate connections of draining and non-draining lymph node to finally end up in the blood and in distant skin, causing new lesions.

IL-4Rα-Expressing B Cells Are Required for CXCL13 Production by Fibroblastic Reticular Cells.

Adaptive type 2 immune responses against the intestinal helminth Heligmosomoides polygyrus (Hp) require the interaction of follicle-associated CXCR5+ dendritic cells with naive T cells in the draining mesenteric lymph nodes (mLNs). However, the source of CXCL13 responsible for attracting CXCR5+ dendritic cells has remained unclear. Using multiplex imaging combined with deep tissue analysis, we observed new CXCL13+ fibroblastic reticular cells surrounding paracortical and cortical B cell follicles in the mLNs of infected mice. CXCL13+ fibroblasts expressed markers of marginal reticular cells (MRCs), and their expansion required lymphotoxin (LT)-dependent interactions between IL-4Rα-expressing B cells and CCL19+ fibroblasts. Infection-induced follicles did not necessarily contain follicular dendritic cells (FDCs), indicating that CXCL13+ fibroblasts may instead drive their formation. These data reveal a role for lymphotoxin signaling to CCL19+ fibroblasts in the development of CXCL13+ MRC-like cells and adaptive type 2 immunity in response to helminth infection.

Development of Sample-Adaptable Holders for Lightsheet Microscopy.

Multi-user core microscopy facilities are often faced with the challenge to adapt or modify existing instruments. This is essential in order to fulfill the requirements of the user community, who wants to image a wide range of model organisms with varying stains and sample thicknesses. In recent years, lightsheet microscopy has turned into an invaluable tool for both live and cleared sample imaging of many different specimens. This brought up new challenges in terms of sample mounting as the classical approach of attachment onto a coverslip cannot be universally applied. Here we describe the development of a diversified holder which extends the range of samples which can be imaged on a Zeiss Lightsheet microscope Z1. We focus on mounting strategies of cleared specimens; however, the holder and mounting strategy can be applied to live specimens too. The proposed methodology provides very high flexibility along with numerous possibilities for adaptation based on imaging specimen size, condition and available clearing reagents. Moreover, the described mounting strategies can be applied to other light sheet microscopes that can mount 1 mL syringes.

FIBCD1 Binds Aspergillus fumigatus and Regulates Lung Epithelial Response to Cell Wall Components.

Aspergillus fumigatus (A. fumigatus) is a ubiquitous fungus of clinical importance associated with development of various pulmonary diseases and allergic hypersensitivity reactions. It is protected against environmental stress by a cell wall that contains polysaccharides such as chitin. We previously demonstrated that fibrinogen C domain-containing protein 1 (FIBCD1) is a membrane-bound protein that binds chitin through a conserved S1 binding site and is expressed in intestinal epithelium and salivary glands. Here, we further localized FIBCD1 protein expression at the surface of bronchial and alveolar human lung epithelium, observed recognition of A. fumigatus cell wall with S1 site-independent recognition. We observed FIBCD1-mediated suppression of IL-8 secretion, mucin production, and transcription of genes associated with airway inflammation and homeostasis in FIBCD1-transfected lung epithelial cells. These modulations were generally enforced by stimulation with A. fumigatus cell wall polysaccharides. In parallel, we demonstrated a FIBCD1-mediated modulation of IL-8 secretion induced by TLR2,-4, and -5. Collectively, our findings support FIBCD1 as a human lung epithelial pattern recognition receptor that recognizes the complex A. fumigatus cell wall polysaccharides and modulates the lung epithelial inflammatory response by suppressing inflammatory mediators and mucins.

Interactions between fibroblastic reticular cells and B cells promote mesenteric lymph node lymphangiogenesis.

Lymphatic growth (lymphangiogenesis) within lymph nodes functions to promote dendritic cell entry and effector lymphocyte egress in response to infection or inflammation. Here we demonstrate a crucial role for lymphotoxin-beta receptor (LTßR) signaling to fibroblastic reticular cells (FRCs) by lymphotoxin-expressing B cells in driving mesenteric lymph node lymphangiogenesis following helminth infection. LTßR ligation on fibroblastic reticular cells leads to the production of B-cell-activating factor (BAFF), which synergized with interleukin-4 (IL-4) to promote the production of the lymphangiogenic factors, vascular endothelial growth factors (VEGF)-A and VEGF-C, by B cells. In addition, the BAFF-IL-4 synergy augments expression of lymphotoxin by antigen-activated B cells, promoting further B cell-fibroblastic reticular cell interactions. These results underlie the importance of lymphotoxin-dependent B cell-FRC cross talk in driving the expansion of lymphatic networks that function to promote and maintain immune responsiveness.The growth of lymph nodes in response to infection requires lymphangiogenesis. Dubey et al. show that the mesenteric lymph node lymphangiogenesis upon helminth infection depends on the signaling loop between the B and fibroblastic reticular cells (FRCs), whereby the FRCs respond to lymphotoxin secreted by B cells by releasing B cell activating factor.

Lymphotoxin-Dependent B Cell-FRC Crosstalk Promotes De Novo Follicle Formation and Antibody Production following Intestinal Helminth Infection.

Secondary lymphoid tissues provide specialized niches for the initiation of adaptive immune responses and undergo a remarkable expansion in response to inflammatory stimuli. Although the formation of B cell follicles was previously thought to be restricted to the postnatal period, we observed that the draining mesenteric lymph nodes (mLN) of helminth-infected mice form an extensive number of new, centrally located, B cell follicles in response to IL-4Rα-dependent inflammation. IL-4Rα signaling promoted LTα1ß2 (lymphotoxin) expression by B cells, which then interacted with CCL19 positive stromal cells to promote lymphoid enlargement and the formation of germinal center containing B cell follicles. Importantly, de novo follicle formation functioned to promote both total and parasite-specific antibody production. These data reveal a role for type 2 inflammation in promoting stromal cell remodeling and de novo follicle formation by promoting B cell-stromal cell crosstalk.

The Intestinal Microbiota Contributes to the Ability of Helminths to Modulate Allergic Inflammation.

Intestinal helminths are potent regulators of their host's immune system and can ameliorate inflammatory diseases such as allergic asthma. In the present study we have assessed whether this anti-inflammatory activity was purely intrinsic to helminths, or whether it also involved crosstalk with the local microbiota. We report that chronic infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb) altered the intestinal habitat, allowing increased short chain fatty acid (SCFA) production. Transfer of the Hpb-modified microbiota alone was sufficient to mediate protection against allergic asthma. The helminth-induced anti-inflammatory cytokine secretion and regulatory T cell suppressor activity that mediated the protection required the G protein-coupled receptor (GPR)-41. A similar alteration in the metabolic potential of intestinal bacterial communities was observed with diverse parasitic and host species, suggesting that this represents an evolutionary conserved mechanism of host-microbe-helminth interactions.

Parasite Proximity Drives the Expansion of Regulatory T Cells in Peyer's Patches following Intestinal Helminth Infection.

Helminth infections are typically chronic in nature; however, the exact molecular mechanisms by which these parasites promote or thwart host immunity remain unclear. Worm expulsion requires the differentiation of CD4(+) T cells into Th2 cells, while regulatory T cells (Tregs) act to dampen the extent of the Th2 response. Priming of T cells requires drainage or capture of antigens within lymphoid tissues, and in the case of intestinal helminths, such sites include the mucosa-associated Peyer's patches (PPs) and the draining mesenteric lymph nodes (MLN). To gain insight into when and where the activation of the adaptive T cell response takes place following intestinal helminth infection, we analyzed Th2 and Treg responses in the PPs and MLN following infection with the murine intestinal helminth Heligmosomoides polygyrus bakeri. Protective Th2 responses were observed to be largely restricted to the MLN, while a greater expansion of Tregs occurred within the PPs. Interestingly, those PPs that formed a contact with the parasite showed the greatest degree of Treg expansion and no evidence of type 2 cytokine production, indicating that the parasite may secrete products that act in a local manner to selectively promote Treg expansion. This view was supported by the finding that H. polygyrus bakeri larvae could promote Treg proliferation in vitro. Taken together, these data indicate that different degrees of Treg expansion and type 2 cytokine production occur within the PPs and MLN following infection with the intestinal helminth H. polygyrus bakeri and indicate that these organs exhibit differential responses following infection with intestinal helminths.

Chitin enhances serum IgE in Aspergillus fumigatus induced allergy in mice.

Aspergillus fumigatus (A. fumigatus) is a ubiquitous fungus that activates, suppresses or modulates the immune response by changing its cell wall structure and by secreting proteases. In this study, we show that chitin acts as an adjuvant in a murine model of A. fumigatus protease induced allergy. The mice were immunised intraperitoneally with A. fumigatus culture filtrate antigen either with or without chitin and were subsequently challenged with the culture filtrate antigen intranasally. Alum was used as an adjuvant control. Compared to alum, chitin induced a weaker inflammatory response in the lungs, measured as the total cell efflux in BAL, EPO and chitinase production. However, chitin enhanced the total IgE, specific IgE and specific IgG1 production as efficiently as alum. Pre-treatment with chitin but not with alum depressed the concentration of the Th2 cytokines IL-4 and IL-13 in BAL fluid. These results shows that chitin, in spite of a reduction of the Th2 cytokine levels in the lungs, enhanced the total and specific IgE production in A. fumigatus culture filtrate induced allergy.

Induction of innate immunity by Aspergillus fumigatus cell wall polysaccharides is enhanced by the composite presentation of chitin and beta-glucan.

Chitin and ß-glucan are conserved throughout evolution in the fungal cell wall and are the most common polysaccharides in fungal species. Together, these two polysaccharides form a structural scaffold that is essential for the survival of the fungus. In the present study, we demonstrated that Aspergillus fumigatus alkali-insoluble cell wall fragments (AIF), composed of chitin linked covalently to ß-glucan, induced enhanced immune responses when compared with individual cell wall polysaccharides. Intranasal administration of AIF induced eosinophil and neutrophil recruitment, chitinase activity, TNF-α and TSLP production in mice lungs. Selective destruction of chitin or ß-glucan from AIF significantly reduced eosinophil and neutrophil recruitment as well as chitinase activity and cytokine expression by macrophages, indicating the synergistic effect of the cell wall polysaccharides when presented together as a composite PAMP. We also showed that these cell wall polysaccharides induced chitin-specific IgM in mouse serum. Our in vivo and in vitro data indicate that chitin and ß-glucan play important roles in activating innate immunity when presented as composite cell wall PAMPs.