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. 2015 Jan 28;10(1):e0117825.
doi: 10.1371/journal.pone.0117825. eCollection 2015.

Milk-based nutraceutical for treating autoimmune arthritis via the stimulation of IL-10- and TGF-β-producing CD39+ regulatory T cells

Massimo Maddaloni  1 , Irina Kochetkova  2 , SangMu Jun  1 , Gayle Callis  2 , Theresa Thornburg  2 , David W Pascual  1
Affiliations

Milk-based nutraceutical for treating autoimmune arthritis via the stimulation of IL-10- and TGF-β-producing CD39+ regulatory T cells

Massimo Maddaloni et al. PLoS One. .

Abstract

Autoimmune diseases arise from the loss of tolerance to self, and because the etiologies of such diseases are largely unknown, symptomatic treatments rely on anti-inflammatory and analgesic agents. Tolerogenic treatments that can reverse disease are preferred, but again, often thwarted by not knowing the responsible auto-antigens (auto-Ags). Hence, a viable alternative to stimulating regulatory T cells (Tregs) is to induce bystander tolerance. Colonization factor antigen I (CFA/I) has been shown to evoke bystander immunity and to hasten Ag-specific Treg development independent of auto-Ag. To translate in treating human autoimmune diseases, the food-based Lactococcus was engineered to express CFA/I fimbriae, and Lactococcus-CFA/I fermented milk fed to arthritic mice proved highly efficacious. Protection occurred via CD39+ Tregs producing TGF-β and IL-10 to potently suppress TNF-α production and neutrophil influx into the joints. Thus, these data demonstrate the feasibility of oral nutraceuticals for treating arthritis, and potency of protection against arthritis was improved relative to that obtained with Salmonella-CFA/I.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Expression of E. coli cfaI fimbrial operon in Lactococcus lactis.
(A) Schematic map for expression of E. coli cfaI in L. lactis (pBzMM153). The elements of the composite promoter are boxed. Each structural gene is fused in-frame to a different lactococcal secretion signal peptide, and each gene is followed by its own STOP codon. No other functional elements are interspersed except for one Shine-Dalgarno (SD) sequence upstream of a fusion between extracellular (Exp4) protein and cfaA. All (SD) sequences are marked. (B) CFA/I fimbriae expression in L. lactis. Lane1, molecular weight (MW) markers; lane 2, Salmonella-CFA/I (H696) strain; lane 3, L. lactis bearing the empty pMSP3535H3 vector; lanes 4,5, pBzMM156 (nisin-inducible promoter) clones 1 and 2; lanes 6,7, pBzMM155 (p23 promoter) clones 1 and 2; and lanes 8–10, pBzMM153 (synthetic composite promoter) clones 1–3. (C, D) Immunogold staining of (C) L. lactis vector and (D) L. lactis-CFA/I with anti-CFA/I antibody. Arrows point to gold particles on fimbrial structures projecting from the cell wall.
Fig 2
Fig 2. L. lactis-CFA/I, not L. lactis vector, is a potent therapeutic for collagen-induced arthritis (CIA).
C57BL/6 males (n = 5/group) were CII-challenged on day 0, and treated orally with 5×108 CFUs L. lactis-CFA/I (L. lactis-pBzMM153) or vector control (L. lactis-pMSP3535H3) in sterile PBS or with vehicle alone on days (A) 14, 21, 28 or (B) on days 18 and 25 post-CIA induction as diagramed. Bacteria were grown in synthetic M17 medium supplemented with 0.5% glucose. A representative example of 6 experiments (n = 5/group) is depicted; * p < 0.01; ✢ p < 0.05 as compared to each control group. (C) Joint pathology was evaluated from decalcified knees from mice in each treatment group (n = 5/group). Representative examples of mid-sagittal knee joint sections are stained with H&E (top row) or toluidine blue (bottom row) from mice treated with two doses of L. lactis-CFA/I (B) upon termination of the study.
Fig 3
Fig 3. L. lactis-CFA/I confers protection against CIA via reduction of neutrophil influx into joints with concomitant reduction in TNF-α, but only slightly reduces anti-CII Ab titers.
Reduced tissue destruction in Fig. 2 is attributed to diminished neutrophil infiltration of the joints (A, B) in L. lactis-CFA/I-treated mice (as described in Fig. 2B). Cell suspensions were analyzed by flow cytometry for Ly-6G+Ly-6C+CD11b+ neutrophils; * p < 0.001 compared with control groups (n = 5/group). (C) L. lactis-CFA/I fails to elicit serum IgG and IgG subclass anti-CFA/I fimbriae and (D) modestly reduces IgG1 and IgG2a anti-CII antibody titers (day 35 post-CIA induction) in orally treated mice from Fig. 2; n = 5 mice/group. (E) LN lymphocytes from L. lactis-CFA/I-treated mice from Fig. 2B (n = 5 mice/group) were CII-restimulated in vitro and showed reduced TNF-α production; * p < 0.001 compared to each control group.
Fig 4
Fig 4. L. lactis-CFA/I reduces CII-specific inflammatory CD4+ T cells with concomitant increases in anti-inflammatory cytokines.
On day 40 post-CIA induction, purified LN CD4+ T cells from each group were restimulated with 50 μg/ml CII for 4 days in presence of syngenic irradiated Ag-presenting cells. (A) L. lactis-CFA/I, not L. lactis vector, suppress IL-6, IL-17, and IFN-γ responses and show elevated (B) TGF-β and IL-10. Depicted are the means ± SD of triplicate cultures as assessed by cytokine-specific ELISA; one of 3 experiments (5 mice/group) is shown. (C) Frequency of CD39+CD4+ T cells in LNs of control and protected mice (5 mice/group). (D, E) Expression of TGF-β for gated LN CD39+CD4+ T cells mice treated with PBS, L. lactis vector, or L. lactis-CFA/I. Representative FACS plots (D) and frequencies of TGF-β+CD39+CD4+ T cells (E) are depicted. For all studies, *p < 0.001, **p < 0.005, and ***p < 0.05 as compared with PBS- and L. lactis vector-treated mice.
Fig 5
Fig 5. L. lactis-CFA/I-induced CD39+CD4+ T cells protect against CIA.
Donor mice were treated with L. lactis-CFA/I or L. lactis vector on days 0, 2, and 4. Three days after the last dose, spleens and LNs were harvested for cell-sorting of CD39-CD4+ and CD39+CD4+ T cells, and adoptively transferred into recipients previously CII-challenged 14 days earlier (n = 6–10 mice/group). Severity and incidence of disease in CIA recipients of (A) L. lactis vector- and (B) L. lactis-CFA/I-induced CD39-CD4+ and CD39+CD4+ T cells are shown; *p < 0.005; ✢ p < 0.01 as compared with PBS- treated mice and with CD39-CD4+ T cell recipients.
Fig 6
Fig 6. Two doses of L. lactis-CFA/I fermented milk protect against CIA.
Groups of C57BL/6 mice (n = 8/group) were CII-challenged on day 0, and treated twice on days 18 and 22 with (A) L. lactis-CFA/I- or L. lactis vector-fermented milk that contained 2.5×108 CFUs or treated with sterile PBS. Mice were monitored for disease until day 39 measuring (B) average clinical score (C) and incidence of arthritis. Arrows indicate days of fermented milk administration; * p ≤ 0.001 as compared to each control group. (D-G) LN CD4+ T cells isolated from each treatment group were restimulated with 50 μg CII in the presence of mitomycin C-treated Ag-presenting cells for 4 days. CIA mice treated with L. lactis-CFA/I fermented milk showed reduced LN (D) IFN-γ and (E) IL-17 production with concomitant increases in (F) IL-10 and (G) TGF-β; * p ≤ 0.001, ** p < 0.05 versus PBS-treated mice; and + p ≤ 0.001 versus L. lactis vector-treated mice.
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