Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Nov 16:8:1575.
doi: 10.3389/fimmu.2017.01575. eCollection 2017.

Toll-Like Receptor 4, but Not Neutrophil Extracellular Traps, Promote IFN Type I Expression to Enhance Th2 Responses to Nippostrongylus brasiliensis

Christophe Pellefigues  1 , Shiau-Choot Tang  1 , Alfonso Schmidt  1 , Ruby F White  1 , Olivier Lamiable  1 , Lisa M Connor  1 , Christiane Ruedl  2 , Jurek Dobrucki  3 , Graham Le Gros  1 , Franca Ronchese  1
Affiliations

Toll-Like Receptor 4, but Not Neutrophil Extracellular Traps, Promote IFN Type I Expression to Enhance Th2 Responses to Nippostrongylus brasiliensis

Christophe Pellefigues et al. Front Immunol. .

Abstract

The induction of Th2 responses is thought to be multifactorial, and emerge from specific pathways distinct from those associated with antagonistic antibacterial or antiviral Th1 responses. Here, we show that the recognition of non-viable Nippostrongylus brasiliensis (Nb) in the skin induces a strong recruitment of monocytes and neutrophils and the release of neutrophil extracellular traps (NETs). Nb also activates toll-like receptor 4 (TLR4) signaling with expression of Ifnb transcripts in the skin and the development of an IFN type I signature on helminth antigen-bearing dendritic cells in draining lymph nodes. Co-injection of Nb together with about 10,000 Gram-negative bacteria amplified this TLR4-dependent but NET-independent IFN type I response and enhanced the development of Th2 responses. Thus, a limited activation of antibacterial signaling pathways is able to boost antihelminthic responses, suggesting a role for bacterial sensing in the optimal induction of Th2 immunity.

Keywords: IFN-I; Nippostrongylus brasiliensis; Th2 response; dendritic cells; helminth; neutrophil extracellular traps; skin immunity; toll-like receptor 4.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Nippostrongylus brasiliensis (Nb) induces the recruitment and activation of neutrophils in the skin. C57BL/6 mice were injected intradermally into the ear with 600 AF488+ Nb or PBS. The resulting inflammatory response in blood and skin was analyzed by flow cytometry at the indicated time points. Each symbol represents one mouse. (A) Proportion of neutrophils (CD11bhi Ly6G+) in peripheral blood leukocytes (PBL). Data are pooled from two independent experiments. (B) Recruitment of leukocyte populations in the ear skin at 24 h. Leukocytes were identified as CD45+ cells, neutrophils and monocytes were identified according to the gating in panel C. Data are from one of three experiments that gave similar results. (C) Representative dot plots depicting the gating of ear skin CD45+ populations expressing Ly6G (neutrophils), high Ly6C (monocytes), and AF488 as a measure of Nb uptake. (D) Pie chart showing the relative proportion of AF488+ populations in skin, identified as in (C). Monocyte-derived DCs (moDCs) are defined as Ly6G− Ly6Chi CD11bhi CD11c+ MHCII+, and CD11b+ dendritic cells (DCs) are Ly6G− Ly6C− CD11c+ MHCII+ CD11b+ CD326−. Data are from one of three experiments that gave similar results. Bar graphs show mean ± SEM. Statistical analyses used the Mann–Whitney test. NS: not significant; *p < 0.05; **p < 0.01; ****p < 0.0001. (B) Symbols close to the legend indicate a comparison of the same population between the two groups.
Figure 2
Figure 2
Nippostrongylus brasiliensis (Nb) induces the formation of neutrophil extracellular traps in skin. C57BL/6 mice were injected intradermally into the ear with 150 AF488+ Nb, and z-stacks of whole-mount ear dermis were analyzed by immunofluorescence and confocal microscopy at different time points. Images are representative of at least two independent experiments. (A) Dermis at 1 h after AF488+ Nb injection showing DAPI+ nuclei (blue) and AF488+ Nb (magenta). Bar = 200 µm. (B) Dermis at 2 h after AF488+ Nb injection showing DAPI+ nuclei (blue), AF488+ Nb (magenta), and Ly6G+ neutrophils (Green). Bar = 50 µm. (C) Dermis at 24 h after AF488+ Nb injection showing DAPI+ nuclei (blue), AF488+ Nb (magenta), myeloperoxidase (Red), and neutrophil elastase (Green). Bar = 50 µm. (D) Dermis at 48 h after AF488+ Nb injection showing DAPI+ nuclei (blue), autofluorescent Nb (magenta), histone H3 citrullination (Green), and neutrophil elastase (Red). Bar = 200 µm.
Figure 3
Figure 3
Toll-like receptor 4 (TLR4) expression is necessary for Nippostrongylus brasiliensis (Nb)-dependent BST2 expression in vitro. Bone marrow (BM) cells were harvested from C57BL/6 mice or the indicated strains, stimulated overnight in the described conditions, and analyzed by flow cytometry. (A) Identification of neutrophils (CD11bhi Ly6G+) and monocytes (CD11bhi Ly6G− Ly6C+) and their uptake of AF488 and expression of BST2 after stimulation with AF488+ Nb (red) or medium (black). (B) BST2 expression on monocytes from BM cultures that were stimulated with AF488+ Nb in the presence of IFNAR1-blocking antibodies or isotype control. (C) BST2 expression on monocytes from BM cultures stimulated with Nb, Nb supernatant (SN), house dust mite (HDM) or Poly(I:C) in the presence of IFNAR1-blocking antibodies or mIgG1. Data are from one of at least two independent experiments that gave similar results; each dot corresponds to a BM culture from a separate mouse. (D) Stimulation of TLR reporter activity by Nb. Cell lines expressing the indicated TLR and their respective controls were cocultured overnight with Nb or no stimulus, and TLR reporter activity was quantified using a colorimetric assay. Reporter activity for each cell line is expressed as fold-change (FC) of the readings for stimulated versus unstimulated cultures. Each dot corresponds to an independent experiment and is the average of triplicate cultures. (E) BM cells from C57BL/6 (WT), TLR4 KO or TLR2 KO mice were cultured with Nb or Poly(I:C) or no stimulus, in the presence of IFNAR1-blocking antibodies or isotype control. BST2 expression on monocytes was assessed after overnight culture. Data are from one of at least two independent experiments that gave similar results; each dot corresponds to a BM culture from a separate mouse. Statistical analyses used the ANOVA with Tukey’s multiple comparisons test. NS: not significant; **p < 0.01, ***p < 0.001, ****p < 0.0001. Symbols above individual bars refer to the p value of the indicated group versus its control condition, which was either unstimulated (C) or WT (E).
Figure 4
Figure 4
Nippostrongylus brasiliensis (Nb) induces a toll-like receptor 4 (TLR4)-dependent expression of IFN-I in the skin. C57BL/6 (WT) or TLR4 KO mice were injected intradermally into the ear with 600 AF488+ Nb. Expression of IFN-I transcripts and of the IFN-I-dependent marker BST2 were examined in ear tissue as indicated. Each dot corresponds to one mouse. (A) Representative flow cytometry contour plots depicting AF488 uptake and expression of BST2 on CD45+ skin populations 24 h after PBS injection, or co-injection of AF488+ Nb together with IFNAR1-blocking antibodies or isotype control. Cell populations were identified as in Figure 1. Fluorescence Minus One (FMO) for BST2 is shown as a control. (B) Expression of BST2 on AF488− and AF488+ skin populations defined as in (A). Bar graphs show mean ± SEM from one of two independent experiments that gave similar results. (C) Quantitative Reverse Transcription PCR for Ifnb1, Ifna (all species) and Ifnk in ear tissue 2 h after Nb injection. Bar graphs show mean ± SEM from two independent experiments each with three mice/group. Statistical analyses used the ANOVA with Tukey’s multiple comparisons test in (B), or Kruskal–Wallis test with Dunn’s multiple comparison in (C) as the distribution of the data could not be considered normal (Shapiro–Wilk normality test). Ns: not significant; *p < 0.05; **p < 0.01.
Figure 5
Figure 5
Toll-like receptor 4 (TLR4), not extracellular DNA, drives the development of an IFN-I signature on LN dendritic cells (DCs) after Nippostrongylus brasiliensis (Nb) injection. C57BL/6 or TLR4 KO mice were injected intradermally into the ear with AF488+ Nb. Uptake of AF488 and expression of CD86 and the IFN-I-dependent marker BST2 were examined in ear draining lymph node (dLN) DCs by flow cytometry 48 h after Nb injection. CD11b+ DCs were CD11c+ MHCIIhi CD11b+ CD326− CD103−. Triple Negative (TN) DCs were CD11c+ MHCIIhi CD11b− CD326− CD103−. Each dot corresponds to one mouse. Data are from one of at least two repeat experiments that gave similar results. (A) AF488 uptake and BST2 expression on subsets of migratory DCs in dLN of C57BL/6 (WT) or TLR4 KO mice after injection of 600 AF488+ Nb. (B) BST2 expression on subsets of migratory DCs in the dLN of C57BL/6 mice after co-injection of 300 Low-Endotoxin (LE, < 5 Endotoxin Units/mL) AF488+ Nb with or without 10,000 formalin-fixed E coli. (C) AF488 uptake and BST2 or CD86 expression on subsets of migratory DCs in the dLN of C57BL/6 mice after injection of 600 AF488+ Nb with or without DNase I treatment. Bar graphs show mean ± SEM. Statistical analyses used the ANOVA with Tukey’s multiple comparisons test. Ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 6
Figure 6
Toll-like receptor 4 (TLR4) enhances the development of CD4+IL4+ T cells after Nippostrongylus brasiliensis (Nb) injection. C57BL/6 or TLR4 KO mice were injected intradermally into the ear with 150 Nb from different preparations. T cell responses were measured in the draining lymph node by intracellular cytokine staining and flow cytometry 7 days after Nb injection. Each dot corresponds to one mouse. Data are from one of at least two repeat experiments that gave similar results. (A) T cell cytokine response in C57BL/6 mice injected with Nb, or Low Endotoxin (LE, <5 Endotoxin U/mL) Nb, or Low Endotoxin Nb preparations (LE-Nb) plus 10,000 formalin-fixed E coli. (B) T cell cytokine response in C57BL/6 (WT) and TLR4 KO mice injected with Nb together with IFNAR1-blocking antibodies or isotype control. Bar graphs show mean ± SEM. Statistical analyses used the ANOVA with Tukey’s multiple comparisons test. Ns: not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Walsh KP, Mills KHG. Dendritic cells and other innate determinants of T helper cell polarisation. Trends Immunol (2013) 34:521–30.10.1016/j.it.2013.07.006 - DOI - PubMed
    1. Fitzsimmons CM, Falcone FH, Dunne DW. Helminth allergens, parasite-specific IgE, and its protective role in human immunity. Front Immunol (2014) 5:61.10.3389/fimmu.2014.00061 - DOI - PMC - PubMed
    1. Palm NW, Rosenstein RK, Medzhitov R. Allergic host defences. Nature (2012) 484:465–72.10.1038/nature11047 - DOI - PMC - PubMed
    1. Méndez-Samperio P. Molecular events by which dendritic cells promote Th2 immune protection in helmith infection. Infect Dis (Auckl) (2016) 4235:1–6.10.1080/23744235.2016.1194529 - DOI - PubMed
    1. Vannella KM, Ramalingam TR, Borthwick LA, Barron L, Hart KM, Thompson RW, et al. Combinatorial targeting of TSLP, IL-25, and IL-33 in type 2 cytokine – driven inflammation and fibrosis. Sci Transl Med (2016) 8:1–13.10.1126/scitranslmed.aaf1938 - DOI - PubMed

LinkOut - more resources