Epithelial-Cell-Derived Phospholipase A2 Group 1B Is an Endogenous Anthelmintic.

Immunity to intestinal helminth infections has been well studied, but the mechanism of helminth killing prior to expulsion remains unclear. Here we identify epithelial-cell-derived phospholipase A2 group 1B (PLA2g1B) as a host-derived endogenous anthelmintic. PLA2g1B is elevated in resistant mice and is responsible for killing tissue-embedded larvae. Despite comparable activities of other essential type-2-dependent immune mechanisms, Pla2g1b-/- mice failed to expel the intestinal helminths Heligmosomoides polygyrus or Nippostrongylus brasiliensis. Expression of Pla2g1b by epithelial cells was dependent upon intestinal microbiota, adaptive immunity, and common-gamma chain-dependent signaling. Notably, Pla2g1b was downregulated in susceptible mice and inhibited by IL-4R-signaling in vitro, uncoupling parasite killing from expulsion mechanisms. Resistance was restored in Pla2g1b-/- mice by treating infective H. polygyrus L3 larvae with PLA2g1B, which reduced larval phospholipid abundance. These findings uncover epithelial-cell-derived Pla2g1b as an essential mediator of helminth killing, highlighting a previously overlooked mechanism of anti-helminth immunity

Transcriptomics identified a critical role for Th2 cell-intrinsic miR-155 in mediating allergy and antihelminth immunity

Allergic diseases, orchestrated by hyperactive CD4^(+) Th2 cells, are some of the most common global chronic diseases. Therapeutic intervention relies upon broad-scale corticosteroids with indiscriminate impact. To identify targets in pathogenic Th2 cells, we took a comprehensive approach to identify the microRNA (miRNA) and mRNA transcriptome of highly purified cytokine-expressing Th1, Th2, Th9, Th17, and Treg cells both generated in vitro and isolated ex vivo from allergy, infection, and autoimmune disease models. We report here that distinct regulatory miRNA networks operate to regulate Th2 cells in house dust mite-allergic or helminth-infected animals and in vitro Th2 cells, which are distinguishable from other T cells. We validated several miRNA (miR) candidates (miR-15a, miR-20b, miR-146a, miR-155, and miR-200c), which targeted a suite of dynamically regulated genes in Th2 cells. Through in-depth studies using miR-155^(−/−) or miR-146a^(−/−) T cells, we identified that T-cell–intrinsic miR-155 was required for type-2 immunity, in part through regulation of S1pr1, whereas T-cell–intrinsic miR-146a was required to prevent overt Th1/Th17 skewing. These data identify miR-155, but not miR-146a, as a potential therapeutic target to alleviate Th2-medited inflammation and allergy

miR-182 and miR-10a Are Key Regulators of Treg Specialisation and Stability during Schistosome and Leishmania-associated Inflammation

A diverse suite of effector immune responses provide protection against various pathogens. However, the array of effector responses must be immunologically regulated to limit pathogen- and immune-associated damage. CD4+Foxp3+ regulatory T cells (Treg) calibrate immune responses; however, how Treg cells adapt to control different effector responses is unclear. To investigate the molecular mechanism of Treg diversity we used whole genome expression profiling and next generation small RNA sequencing of Treg cells isolated from type-1 or type-2 inflamed tissue following Leishmania major or Schistosoma mansoni infection, respectively. In-silico analyses identified two miRNA “regulatory hubs” miR-10a and miR-182 as critical miRNAs in Th1- or Th2-associated Treg cells, respectively. Functionally and mechanistically, in-vitro and in-vivo systems identified that an IL-12/IFNγ axis regulated miR-10a and its putative transcription factor, Creb. Importantly, reduced miR-10a in Th1-associated Treg cells was critical for Treg function and controlled a suite of genes preventing IFNγ production. In contrast, IL-4 regulated miR-182 and cMaf in Th2-associed Treg cells, which mitigated IL-2 secretion, in part through repression of IL2-promoting genes. Together, this study indicates that CD4+Foxp3+ cells can be shaped by local environmental factors, which orchestrate distinct miRNA pathways preserving Treg stability and suppressor function

IFNγ and IL-12 restrict Th2 responses during Helminth/Plasmodium co-infection and promote IFNγ from Th2 cells

Parasitic helminths establish chronic infections in mammalian hosts. Helminth/Plasmodium co-infections occur frequently in endemic areas. However, it is unclear whether Plasmodium infections compromise anti-helminth immunity, contributing to the chronicity of infection. Immunity to Plasmodium or helminths requires divergent CD4+ T cell-driven responses, dominated by IFNγ or IL-4, respectively. Recent literature has indicated that Th cells, including Th2 cells, have phenotypic plasticity with the ability to produce non-lineage associated cytokines. Whether such plasticity occurs during co-infection is unclear. In this study, we observed reduced anti-helminth Th2 cell responses and compromised anti-helminth immunity during Heligmosomoides polygyrus and Plasmodium chabaudi co-infection. Using newly established triple cytokine reporter mice (Il4gfpIfngyfpIl17aFP635), we demonstrated that Il4gfp+ Th2 cells purified from in vitro cultures or isolated ex vivo from helminth-infected mice up-regulated IFNγ following adoptive transfer into Rag1-/- mice infected with P. chabaudi. Functionally, Th2 cells that up-regulated IFNγ were transcriptionally re-wired and protected recipient mice from high parasitemia. Mechanistically, TCR stimulation and responsiveness to IL-12 and IFNγ, but not type I IFN, was required for optimal IFNγ production by Th2 cells. Finally, blockade of IL-12 and IFNγ during co-infection partially preserved anti-helminth Th2 responses. In summary, this study demonstrates that Th2 cells retain substantial plasticity with the ability to produce IFNγ during Plasmodium infection. Consequently, co-infection with Plasmodium spp. may contribute to the chronicity of helminth infection by reducing anti-helminth Th2 cells and converting them into IFNγ-secreting cells

Antibody feeding of IFITM expressing cells.

<p>Live A549-IFITM-HA cells were incubated with 5 µg/ml anti-HA at 37°C to allow endocytosis of bound antibody molecules. Subsequently, the cells were washed, fixed, permeabilised and incubated with an anti-rat Alexa-488 conjugate. A) Control A549 cells show no specific staining. B) In IFITM1-HA cells the majority of labelling is at the plasma membrane. C) IFITM3-HA cells that were not incubated with anti-HA antibody show no labelling. For IFITM2-HA and IFITM3-HA expressing cells (D and E) the majority of cells are not labelled, however in both cases a minority of cells do show punctate labelling indicative of IFITM-mediated internalisation of anti-HA antibodies. In D and E, the boxed region has been enlarged. All images were taken using the same microscope settings and adjusted uniformly. Scale bars represent 15 µm.</p

Analysis of IFITM NTD antibodies.

<p>Two commercially available antibodies targeting either the IFITM1-NTD or the IFITM3-NTD were screened by western blot to assess specificity using HA-tagged IFITM1-3 (M1, M2 and M3) cell lines along with control A549 cells. Proteins were also identified using the HA epitope. Blots were imaged on a Li-COR Odyssey system that uses far-red fluorophore conjugated secondary antibodies. In the overlay image, red represents anti-IFITM-NTD labelling and green represents anti-HA labelling. A) Anti-IFITM1-NTD detects IFITM1 and shows cross-reactivity with IFITM3. B) Anti-IFITM3-NTD detects IFITM3 and has cross-reactivity with IFITM2. VDAC was used as a loading control.</p

Trypsin cleavage of HA-tagged IFITM1.

<p>A) Predicted trypsin cleavage sites in hu IFITM1 CTD (Model 3, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104341#pone-0104341-g001" target="_blank">Fig. 1</a>). B) IFITM1-HA cells were treated with exogenous trypsin for 5 to 30 mins at 37°C. The trypsin was inactivated with soybean trypsin inhibitor, the cells were lysed and the cellular proteins separated by SDS-PAGE. After transfer, proteins were identified with anti-IFITM1-NTD (i) and anti-HA (ii) antibodies. VDAC was used as a loading control (iii). Control samples were untreated (UN), or treated with SBTI-inactivated trypsin (IN). In the overlay image, red represents anti-IFITM1-NTD labelling and green represents anti-HA labelling (iv).</p

Cellular distribution of the human IFITM proteins.

<p>C-terminal domain HA-tagged IFITM1, IFITM2, IFITM3 and control A549 cell lines were stained intact, or following permeabilisation, with an anti-HA antibody and a secondary anti-rat Alexa-488 antibody. A) Permeabilised A549 cells show no specific staining. B-D) IFITM1-HA, IFITM2-HA and IFITM3-HA have distinct cellular distributions in permeabilised cells. E) Intact A549 cells (no detergent treatment) show no specific staining. F) Intact IFITM1-HA cells show positive cell surface HA staining. G) Intact IFITM2-HA cells show no detectable HA staining. H) Although the majority of intact IFITM3-HA cells show no anti-HA labelling, a minority (<1%) show low-level positive staining. Nuclei were labelled with Hoechst. All images are maximum projections of 0.25 µm optical sections taken through the depth of the cells using a confocal microscope. All images were taken using the same microscope settings and the levels adjusted uniformly. Scale bar represents 15 µm.</p

Co-staining with N- and C-terminal antibodies.

<p>Permeabilised IFITM2-HA (A) and IFITM3-HA (B) expressing cells were stained with antibodies against the C-terminal HA-tag (green [Alexa-488]) and the NTD, using the anti-IFITM3-NTD antibody (red [Alexa-647]). Images are single optical sections (0.25 µm thick) through the cells. Scale bars represent 15 µm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104341#pone-0104341-t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104341#pone-0104341-t003" target="_blank">3</a> for image analysis.</p