Lactobacillus pentosus Modulates Immune Response by Inducing IL-10 Producing Tr1 Cells

Jung Eun Kim; Amit Sharma; Garima Sharma; So Young Lee; Hee Soon Shin; Dipayan Rudra; Sin-Hyeog Im

doi:10.4110/in.2018.19.e39

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Kim, Sharma, Sharma, Lee, Shin, Rudra, and Im: Lactobacillus pentosus Modulates Immune Response by Inducing IL-10 Producing Tr1 Cells

Original Article

Immune Netw 2019;19(6):e39.

Published online: 4 December 2019

DOI: https://doi.org/10.4110/in.2018.19.e39

Lactobacillus pentosus Modulates Immune Response by Inducing IL-10 Producing Tr1 Cells

Jung Eun Kim^1,^†, Amit Sharma^2,^†, Garima Sharma², So Young Lee^3,⁴, Hee Soon Shin^3,⁴, Dipayan Rudra², Sin-Hyeog Im^2,^*

¹SK Biopharmaceuticals (HQ), Seongnam 13494, Korea

²Division of Integrative Biosciences and Biotechnology, Department of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang 37673, Korea

³Korea Food Research Institute, Wanju 55365, Korea

⁴KFRI School, Korea University of Science and Technology, Wanju 55365, Korea

^*Correspondence to Sin-Hyeog Im Division of Integrative Biosciences and Biotechnology, Department of Life Sciences, Pohang University of Science and Technology (POSTECH), 77 Cheongam-ro, Nam-gu, Pohang 37673, Korea. E-mail: iimsh@postech.ac.kr

^† These authors contributed equally to this work.

Conflict of Interest Kim JE is an employee of SK Biopharmaceuticals. Im SH is a major shareholder of Immunobiome Inc. Other authors declare no potential conflicts of interest.

Received 2 November 2019 Revised 15 December 2019 Accepted 17 December 2019

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Several gut commensals have been shown to modulate host immune response. Recently, many food derived microbes have also been reported to affect the immune system. However, a mechanism to identify immunostimulatory and immunoregulatory microbes is needed. Here, we successfully established an in vitro screening system and identified an immunoregulatory bacterium, Lactobacillus pentosus KF340 (LP340), present in various fermented foods. LP340 induced a regulatory phenotype in mice Ag presenting cells which, in turn, induced IL-10 and IFN-γ producing Type 1 regulatory T cells (Tr1 cells) from naïve CD4⁺ T cells. Naïve CD4⁺ T cells co-cultured with LP340 treated dendritic cells highly expressed cytokine receptor IL-27R and were CD49b and lymphocyte-activation gene 3 double positive. Oral administration of LP340 in mice with atopic dermatitis reduced cellular infiltration in affected ear lobes and serum IgE levels, thus, ameliorating the disease symptoms. This suggests a systemic immunoregulatory effect of LP340. These findings demonstrate that LP340, a bacterium derived from food, prevents systemic inflammation through the induction of IL-10 producing Tr1 cells.

Keywords: Tr1 cells, Lactobacillus pentosus, Interleukin-10, IL-10, Atopic dermatitis

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Figure 1.

LP340 preferentially skews the immune microenvironment to immunoregulatory phenotype, both with total mLN cells and APCs. (A-C) Total mLN cells were co-cultured with probiotics and supernatant was used for ELISA, as described in (A) schematics of primary screening method (B) cytokines, IL-10 and IL-12 levels (C) cells were treated with LP340 and LP756 at different concentrations and incubation times followed by measurement of cytokines. (D-F) enriched splenic CD11c⁺ cells were treated with LP340 or LP756 and (D) qRT-PCR analysis of Il10 and Tgfb1 gene expression (E) cytokine analysis by ELISA and (F) flow cytometry analysis of MHCII, CD80, CD86, PD-L1, CD40, ICOS, CD209, and CD103. All data are representative of 3 independent experiments and are presented as mean±SD. NS, not significant; ND, not detectable; ICOS, inducible T-cell costimulator. * p<0.05, ^** p<0.01, ^*** p<0.001, ^**** p<0.0001.

Figure 2.

DCs exposed to LP340 induce CD4⁺ T-cells to produce IL-10. (A-D) Splenic CD11c⁺ cells were co-cultured with LP340 and LP756, then primed CD11c⁺ cells were co-cultured with naïve CD4⁺ T-cells in presence of anti-CD3 and IL-2. Culture supernatants were used for ELISA of cytokines (A) IL-10 (B) IFN-γ, IL-17A, and TGF-β. (C) CD4⁺ T-cells were stimulated with PMA/Ionomycin and used for flow cytometry analysis of cytokines IL-10, IFN-γ, IL-4 and IL-17A (D) CD4⁺ T-cells were also analyzed by flow cytometry for FOXP3 expression and IL-10 expression in CD4⁺ T-cells. (E) CD4⁺ IL-10⁺, and (F) CD4⁺ IFN-γ⁺ IL-10⁺ T-cells were analyzed by flow cytometry. All data are representative of 3 independent experiments and presented as mean±SD. * p<0.05, ^** p<0.01, ^*** p<0.001, ^**** p<0.0001.

Figure 3.

LP340 induce a Tr-1 cell like phenotype in CD4⁺ T-cells. (A-E) Splenic CD11c⁺ cells were co-cultured with LP340 and primed CD11c⁺ cells were co-cultured with naïve CD4⁺ T-cells in presence of anti-CD3 and IL-2. (A) Cells were analyzed for IL-27R and (B) CD49b⁺ LAG-3⁺ expression, gated on total CD4⁺ T-cells. The 4 populations sorted by expression of CD49b & LAG-3 were cultured and cell supernatants were used for (C) IL-10 & IFN-γ cytokine ELISA (D) Heat killed LP340 was used for priming the DCs and IL-10 ELISA was performed with supernatant (E) Splenic CD11c⁺ cells from TLR knock-out mice (TLR1^−/−, TLR2^−/−, TLR3^−/−, TLR4^−/−, TLR5^−/−, and TLR9^−/−) were treated with LP340 and IL-10 cytokine ELISA was performed. Data are representative of 3 (A-D) and 2 (E) independent experiments and presented as mean±SD. IL-27R, IL-27 receptor; ND, not detectable; WT, wild type. * p<0.05, ^** p<0.01, ^*** p<0.001, ^**** p<0.0001.

Figure 4.

LP340 ameliorates HDM induced AD. (A-F) Mice orally fed with PBS or LP340 were immunized with DNCB and subsequently were challenged with HDM as depicted in (A) experimental scheme. (B) Ear thickness was measured till 30 days post induction and (C) gross ear lesions were observed (D) Inflammation and cellular infiltration in ear tissue was analyzed by histology, H&E staining (E) Serum IgE levels were measured by ELISA (F) Mast cell (left panel, black arrowheads) and neutrophil (right panel, yellow arrowheads) infiltration was observed by IHC of ear tissue. Data are representative of 3 independent experiments and presented as mean±SD. * p<0.05, ^** p<0.01, ^**** p<0.0001.

Table 1.

Primer sequences for qRT-PCR

Gene	Primer sequence (5′ → 3′)
Hprt	F	TTATGGACAGGACTGAAAGAC
	R	GCTTTAATGTAATCCAGCAGGT
Cd80	F	ACCCCCAACATAACTGAGTCT
	R	TTCCAACCAAGAGAAGCGAGG
Cd86	F	TGTTTCCGTGGAGACGCAAG
	R	CAGCTCACTCAGGCTTATGTTTT
H2Ab	F	CACTCTGGTCTGTTCGGTGAC
	R	CCTCTCCCTGATGAGGGGTC
Cd274	F	GGAATTGTCTCAGAATGGTC
	R	GTAGTTGCTTCTAGGAAGGAG
Ilt3	F	ATGGGCACAAAAAGAAGGCTAA
	R	CTGTGTCCTGATGCACAACTG
Ido	F	GCTTTGCTCTACCACATCCAC
	R	CAGGCGCTGTAACCTGTGT
Socs3	F	CCCTTGCAGTTCTAAGTTCAACA
	R	ACCTTTGACAAGCGGACTCTC
Cox2	F	TGGCTGCAGAATTGAAAGCCCT
	R	AAAGGTGCTCGGCTTCCAGTAT
Il10	F	ATAACTGCACCCACTTCCCA
	R	TCATTTCCGATAAGGCTTGG
Il27	F	CACCTCCGCTTTCAGGTGC
	R	AGGTATAGAGCAGCTGGGGC
Tgfb1	F	CTCCCGTGGCTTCTAGTGC
	R	GCCTTAGTTTGGACAGGATCTG
Il12p40	F	GGAAGCACGGCAGCAGAATA
	R	AACTTGAGGGAGAAGTAGGAATGG

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