Oral Probiotic Control Skin Inflammation by Acting on Both Effector and Regulatory T Cells

Probiotics are believed to alleviate allergic and inflammatory skin disorders, but their impact on pathogenic effector T cells remains poorly documented. Here we show that oral treatment with the probiotic bacteria L. casei (DN-114 001) alone alleviates antigen-specific skin inflammation mediated by either protein-specific CD4+ T cells or hapten-specific CD8+ T cells. In the model of CD8+ T cell-mediated skin inflammation, which reproduces allergic contact dermatitis in human, inhibition of skin inflammation by L. casei is not due to impaired priming of hapten-specific IFNγ-producing cytolytic CD8+ effector T cells. Alternatively, L. casei treatment reduces the recruitment of CD8+ effector T cells into the skin during the elicitation (i.e. symptomatic) phase of CHS. Inhibition of skin inflammation by L. casei requires MHC class II-restricted CD4+ T cells but not CD1d-restricted NK-T cells. L casei treatment enhanced the frequency of FoxP3+ Treg in the skin and increased the production of IL-10 by CD4+CD25+ regulatory T cells in skin draining lymph nodes of hapten-sensitized mice. These data demonstrate that orally administered L. casei (DN-114 001) efficiently alleviate T cell-mediated skin inflammation without causing immune suppression, via mechanisms that include control of CD8+ effector T cells and involve regulatory CD4+ T cells. L. casei (DN-114 001) may thus represent a probiotic of potential interest for immunomodulation of T cell-mediated allergic skin diseases in human

<i>In vivo</i> suppressive function of CD4⁺CD25⁺ Tregs from <i>L. casei</i>-treated mice.

<p>(A) Naïve C57Bl/6 were treated daily from d-14 to d-1 with either <i>L. casei</i> or NaCl. On d-1, CD4⁺ CD25⁺ T cells were purified from pooled pLN, mLN and spleens of each group of mice and transferred <i>i.v</i> together with naive purified CD8⁺ T cells into naive CD3ε°/° recipients. On day 0, untransferred and transferred CD3ε°/° recipients were DNFB-sensitized and challenged 5 days later with DNFB, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004903#pone-0004903-g001" target="_blank">Fig 1</a> legend. (B) Ear swelling was determined at various time after challenge in CD3ε°/° that were either untransferred (dotted lines) or transferred with CD8⁺ T cells alone (white squares), or with CD8⁺ T cells together with CD4⁺CD25⁺ T cells from <i>L. casei</i>- treated (black circle) or NaCl-treated (empty circles) donors.</p

Increased IL-10 production by CD4⁺CD25⁺ T cells from <i>L. casei</i> treated mice.

<p>C57Bl/6 mice were treated with <i>L. casei</i> or NaCl from Day-14 before DNFB skin sensitization until Day+5 post-sensitization. (A) on Day 5, spleen cell suspensions from <i>L. casei</i>-treated (right panel) or NaCl–treated (<i>left panel</i>) mice were stained for FACS analysis. The dot plot shows the percentages of CD4⁺CD25⁺ cells on gated CD4⁺ T cells. (B) On Day 5, CD4⁺CD25⁺ T cells purified from spleen of <i>L. casei</i> treated (black bar) or NaCl- treated (white bar) mice were restimulated <i>in vitro</i> with soluble anti-mouse CD3+mIL-2 and 72 hr culture supernatants were titrated for IL-10 by Elisa.</p

Effect of <i>L. casei</i> on CD8⁺ effectors and FoxP3⁺ regulatory T cells in the skin.

<p>Total cells were extracted from ears of either <i>L. casei</i> treated or NaCl mice that were either unsensitized, or DNFB-sensitized and harvested at 24 h, 48 h and 72 h post DNFB ear challenge. (A) Dot plot FACS analysis on gated CD45⁺ cells of ear cell suspensions staining for CD8 and granzyme B at 48 hr post challenge. (B) Histograms representation of the percentage of granzyme B⁺ CD8⁺ T cells (black portion) and granzyme B⁻ CD8⁺: white portion) among gated CD45⁺ leucocytes from NaCl- (left) and L. Casei (right) treated mice at various time point after ear challenge. (C) Dot plot FACS analysis of gated CD45⁺×CD4⁺ T cells after ear cell suspensions stained for CD4, CD25 and FoxP3 at 48 hr post DNFB challenge in naïve (left) or sensitized (right) mice, treated with NaCl (top) or <i>L. Casei</i> (bottom). (D) Corresponding ear swelling at 24 h, 48 h and 72 h post- DNFB challenge in DNFB sensitized mice, treated with NaCl (white circles) or <i>L. casei</i> (black circles).</p

Effect of <i>L. casei</i> on hapten-specific CD8⁺ CTL <i>in vivo</i>.

<p>Hapten-specific <i>in vivo</i> CTL assay was carried out in mice treated daily from day -14 with either NaCl (white bars) or <i>L. casei</i> (black bars). Mice were sensitized on day 0 with 0.5% DNFB and 5 days later were injected intravenously with a 1∶1 mixture of DNBS-pulsed and unpulsed spleen cells as target cells, stained with 0.5 µmol/L and 5 µmol/L of CFSE, respectively. Twenty-four hours later, the percentage of stained cells in pooled pLN and spleen was analyzed by FACS. In vivo cytotoxicity was calculated by determining the ratio of control targets/pulsed targets. Results are expressed as mean±SD of the percentage of hapten-specific cytotoxicity.</p

Inhibition of CHS by <i>L. casei</i> requires CD4⁺ T cells.

<p>Aβ°/° mice (A) and CD1d°/° mice (B) were treated daily from Day-14 before DNFB skin sensitization (Day 0) until the end of experiment with either live <i>L. casei</i> (black circle) or NaCl (white circle). All mice were sensitized on day 0 with DNFB and ear challenged on day 5 with DNFB. The CHS response was determined by ear swelling (µm) as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004903#pone-0004903-g001" target="_blank">Fig. 1</a> legend. Statistics (<i>p</i> values) were performed using the Mann Whitney two-sample and Student's <i>t</i>-test. Dotted lines represent ear swelling values after ear challenge of unsensitized mice.</p

Immunomodulatory effect of <i>L. casei</i> on DTH responses.

<p>C57Bl/6 mice were treated daily with either live <i>L. casei</i> (black circle) or physiologic water (white circle) from day –14 before sensitization until the end of the experiment. (A) CHS to DNFB was induced by skin painting on day 0 with 0.5% DNFB and ear challenge on day 5 with 0.15% DNFB (right ear) or the vehicle alone (left ear). The CHS response was determined by ear swelling at various time points after challenge, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004903#s2" target="_blank">Material and Methods</a>. The dotted line represents ear swelling after ear painting with 0.15% DNFB in unsensitized mice. (B) DTH to OVA was induced by subcutaneous injection on day 0 with 50 µg OVA mixed with CFA and footpad challenge 7 days later with either 250 µg aggregated ovalbumin (right footpad) or physiologic water (left footpad). The DTH response was determined by footpad swelling at various time points after challenge, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004903#s2" target="_blank">Material and methods</a>. The dotted line represents foodpad swelling after footpad challenge with OVA in mice immunized with CFA alone. Results are expressed as mean±SD of swelling for 7–10 mice per group. Data are representative of three to ten experiments. <i>p</i> values were performed using the Mann-Whitney two-sample and Student's t-tests.</p

Netrin-1 blockade inhibits tumour growth and EMT features in endometrial cancer

Netrin-1 is upregulated in cancers as a protumoural mechanism1. Here we describe netrin-1 upregulation in a majority of human endometrial carcinomas (ECs) and demonstrate that netrin-1 blockade, using an anti-netrin-1 antibody (NP137), is effective in reduction of tumour progression in an EC mouse model. We next examined the efficacy of NP137, as a first-in-class single agent, in a Phase I trial comprising 14 patients with advanced EC. As best response we observed 8 stable disease (8 out of 14, 57.1%) and 1 objective response as RECIST v.1.1 (partial response, 1 out of 14 (7.1%), 51.16% reduction in target lesions at 6 weeks and up to 54.65% reduction during the following 6 months). To evaluate the NP137 mechanism of action, mouse tumour gene profiling was performed, and we observed, in addition to cell death induction, that NP137 inhibited epithelial-to-mesenchymal transition (EMT). By performing bulk RNA sequencing (RNA-seq), spatial transcriptomics and single-cell RNA-seq on paired pre- and on-treatment biopsies from patients with EC from the NP137 trial, we noted a net reduction in tumour EMT. This was associated with changes in immune infiltrate and increased interactions between cancer cells and the tumour microenvironment. Given the importance of EMT in resistance to current standards of care2, we show in the EC mouse model that a combination of NP137 with carboplatin-paclitaxel outperformed carboplatin-paclitaxel alone. Our results identify netrin-1 blockade as a clinical strategy triggering both tumour debulking and EMT inhibition, thus potentially alleviating resistance to standard treatments