Leukotriene B4 modulation of murine dendritic cells affects adaptive immunity

Accepted Manuscript Title: Leukotriene B4 modulation of murine dendritic cells affects adaptive immunity Authors: Marco Antonio Pires-Lapa, Marianna Mainardi Koga, Ildefonso Alves da Silva- Jr, Luciano Ribeiro Filgueiras, Sonia Jancar PII: DOI: Reference:

S1098-8823(18)30117-5 https://doi.org/10.1016/j.prostaglandins.2019.02.001 PRO 6319

To appear in:

Prostaglandins and Other Lipid Mediators

Received date: Revised date: Accepted date:

21 August 2018 18 January 2019 4 February 2019

Please cite this article as: Pires-Lapa MA, Mainardi Koga M, da Silva- IA, Ribeiro Filgueiras L, Jancar S, Leukotriene B4 modulation of murine dendritic cells affects adaptive immunity, Prostaglandins and Other Lipid Mediators (2019), https://doi.org/10.1016/j.prostaglandins.2019.02.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Leukotriene B4 modulation of murine dendritic cells affects adaptive immunity

Marco Antonio Pires-Lapa1, Marianna Mainardi Koga2*, Ildefonso Alves da Silva-Jr3,

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Luciano Ribeiro Filgueiras4, Sonia Jancar5

Department of Immunology, Institute of Biomedical Sciences, University of São Paulo,

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São Paulo, Brazil.

*Present address: Department of Biochemistry, CIIL, Faculty of Biology and Medicine, University of Lausanne, Epalinges, Switzerland.

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# 1 and 2 contributed equally to the work.

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Email addresses: 1- [email protected]; 2- [email protected]; 3-

*

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[email protected], 4- [email protected]; 5- [email protected]

Corresponding author: Sonia Jancar, Department of Immunology, Institute of

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Biomedical Sciences, University of Sao Paulo, Av. Prof. Lineu Prestes 1730, 05508-

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900, São Paulo, SP, Brazil, [email protected].

Highlights

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Murine BM-DCs produce and respond to LTB4, upregulating BLT-1 expression

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LTB4 stimulation increased CD86 expression in BM-DCs and enhanced their T cell priming activity BM-DCs exposed to LTB4 can favor Th2/Treg adaptive immune responses

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Abstract

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Dendritic cells (DCs) link innate and adaptive immunity. The microenvironment

generated during the innate immunity affects DCs and the type of adaptive immunity generated. Lipid mediators are released early in inflammation and could modify the

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functional state of DCs. Leukotriene B4 (LTB4) has a wide range of effects on

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macrophages and in the present study we investigated if it also affects DCs. Murine

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bone marrow-derived DCs were employed and it was found that stimulation of DCs

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with LTB4 (10 nM) increased the gene expression of the high affinity receptor BLT-1 but not of BLT-2. It also increased the co-stimulatory molecule CD86 expression but

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did not affect CD80 and CD40. LTB4-stimulated DCs acquired the capacity to present antigen to T lymphocytes, evidenced by antigen-specific proliferation of CD4+

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lymphocytes in co-cultures of ovalbumin-loaded DCs with DO11.10 splenocytes. LTB4stimulated DCs induced Treg proliferation and increased Th2 cytokine IL-13 the co-

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cultures. Expression of transcription factor genes, Gata3 and Foxp3 (Th2 and Treg, respectively) were also found increased. However, the expression of Th1 transcription

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factor (Tbet) and Th17 (RorγT) were not affected. These results indicate that LTB4 affects DCs and modulates the type of adaptive immune response.

Key words: Leukotriene B4, dendritic cells, adaptive immunity

Introduction Dendritic cells (DCs) are key players in priming the adaptive immune response. Their ability to sense peripheral information and transmit it to naïve T cells, however, requires a maturation process in which DCs increase the expression of cell-surface molecules like MHC II, CD80, CD86, and CD40. Activation/maturation of DCs can be

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triggered by signals such as microbial patterns, danger signals, and inflammatory cytokines and mediators that also enhance their migratory skills allowing them to go to

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secondary lymphoid organs for the antigen presentation and T cell priming. Then, the T

cell differentiation fate will be determined not only by the antigen presentation itself, but also by the soluble signals secreted by DCs, which are dependent on the ones that are

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sensed when DCs are activated. Thus, micro environmental clues during activation of

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DCs are essential for the T helper response to be developed [1].

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Microbial patterns and inflammatory signals activate phospholipase A2 (PLA2),

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which releases the arachidonic acid esterified in the cell membranes that can be converted to prostaglandins, tromboxanes, leukotrienes, PAF among several other lipid

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mediators of inflammation. The role of these lipids in inflammation and innate immunity has been extensively studied, among them leukotriene (LT) B4 is a potent stimulator of

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inflammation by increasing the expression of MyD88 (myeloid differentiation factor 88) in macrophages, the adaptor molecule for the majority of receptors that belong to the

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toll-like family [2, 3]. LTB4 is a metabolite of arachidonic acid generated by the 5lipoxygenase (5-LO) enzyme. This mediator acts on G-protein coupled receptors present in the membrane of several cell types; the BLT-1 is the LTB4 high affinity

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receptor whereas the BLT-2 is the low affinity receptor [4]. DCs are the professional antigen-presenting cells and at the site of

infection/inflammation DCs are exposed to a plethora of mediators that could affect them and consequently the adaptive immunity. A study from our group has shown that the lipid mediator PAF modulates adaptive immunity in mice by affecting DCs function

[5] There is evidence that DCs express LTB4 receptors [6]. Regarding the role of these receptors expressed by DCs in the type of adaptive immunity induced by antigen is controversial. In experimental models of asthma, BLT1 expressed by DCs is responsible for induction of Th2 response to allergen. Also, migration of DCs to regional lymph nodes in the airways as well as airway hyperresponsiveness, are

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dependent on the LTB4/BLT1 axis [7-9]. In contrast, in an experimental model of inflammatory bowel disease, Zhou et al. [10] observed that activation of BLT1 in DCs

favors a Th1 and Th17 differentiation. This was concluded from experiments using DCs

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from BLT1-deficient mice.

In the present study we aimed to further investigate the effect of LTB4 on murine bone

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marrow-derived DCs, focusing on LTB4 effect on co-stimulatory molecules expression in DCs and on their antigen presenting function. LPS was employed as control stimulus

Material and Methods

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Mice

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for DCs maturation [11].

Eight- to ten-week-old BALB/c male mice were obtained from the Department of

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Immunology Animal Facility at the University of São Paulo, and kept in micro-isolator cages under specific pathogen-free conditions. Experiments were performed following

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the guidelines for animal use and care approved by the Institutional Animal Care and Use Committee at the Institute of Biomedical Sciences of the University of São Paulo

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(CEUA – ICB/USP: n. 044, p. 31, b. 03).

Bone marrow-derived DC (BM-DC) generation and culture Bone marrow-derived DCs (will be called DCs from now on) were derived in vitro from bone marrow cells as previously described [12], with a few modifications. Briefly, bone marrow cells were collected from the femur of BALB/c mice and cultured

in RPMI 1640 (GIBCO, Grand Island, NY, USA) medium supplemented with 10% heatinactivated fetal bovine serum (FBS-GIBCO, GO, Brazil) and an antibiotic-antimycotic solution (GIBCO, Grand Island, NY, USA). Cells were cultured for 6 days in tissue culture dishes (Becton Dickinson, Franklin Lakes, NJ, USA) with 20 ng/mL of recombinant murine granulocyte–macrophage colony-stimulating factor (rmGM-CSF—

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Peprotech, Rocky Hill, NJ, USA). Half of the medium was replaced and rmGM-CSF stimulation renewed on day 3 of culture. On day 6, non-adherent cells were collected by gentle pipetting and transferred to six-well plates (106 cells/mL). Cells were then

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treated with LTB4 (Cayman Chemical, Ann Arbor, MI, USA) (1, 10 or 100 nM) with or

without the addition of LPS (Sigma-Aldritch, Saint Louis, MO, USA) (1 µg/mL) for 24

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hours.

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Real-time RT-PCR

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Total RNA was isolated with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA)

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and cDNA was generated from total RNA using the Revert Aid First Strand cDNA

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Synthesis Kit (Thermo Scientific, Rockford, IL, USA), according to manufacturer’s instructions. Real-time PCR was performed with the StepOnePlus Real-Time PCR System (Applied Biosystems, USA), using SYBR Green (SYBR Green Master Mix,

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Applied Biosystems, Warrington, UK) and specific primers: Ltbr1 (FW, 5’- CATGCCAC

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CAGGAGAAGAAG-3’ and RV, 5’-GGCACTAAGACAGATTCAAGG-3’), Ltbr2 (FW, 5’GCTCAGTAGTGTCTCATTCCC-3’ and

RV,

5’-

GGCTTCCACCTGTCACCTT-3’),

Myd88 (FW, 5’-CTGTAAAGGCTTCTCGGACTC-3’ and RV, 5’-GTGAGGATATACTG

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AAGGAGCTG-3’), Tbet (FW, 5’-CGCTCACTGCTCGGAACTCT-3´ and RV, 5’-TCC TGTCTCCAGCCGTTT CT-3’), Gata3 (FW, 5’-GTCCCCATTAGCGTTCCTC-3’ and RV, 5’-CCTTATCAAGCCCAAGCGAA-3’), Foxp3 (FW, 5’-AGACTGCACCACTTCTCTCT-3’ and RV, 5’-GGTTGTGAGAAGGTCTTCGAG-3’), Rorγt (FW, 5’-GAGGTGCTGGAA GATCTGC-3’ and RV, 5’-TCTGCAAGACTCATCGACAAG-3’), and Hprt (FW, 5’-AAC AAAGTCTGGCCTGTATCC-3’

and

RV,

5’-CCCCAAAATGGTTAAGGTTGC-3’).

Relative gene expression was calculated using the

2-ΔΔCt

method as previously

described [13]. Data were normalized by the housekeeping gene (Hprt) and expressed as fold change relative to control (untreated group).

In vitro lymphocyte proliferation assay DCs were incubated for 24 h with 100 μg/mL of OVA (Albumine Grade V,

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Sigma-Aldrich, Saint Louis, MO, USA) alone or together with LTB4 (1, 10 and 100 nM) or LPS (1 μg/mL) as described above on day 6. On day 7, cells were washed to

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remove free OVA and seeded on 96-well flat-bottom plates. CFSE (Cell Trace™ CFSE

Cell Proliferation Kit-Invitrogen, Eugene, OR, USA)-stained BALB/c DO11.10 total

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splenocytes were co-cultured with DCs in a 1:4 ratio (DCs: splenocytes) for 3 days.

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FACS analysis

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Fluorochrome-conjugated antibodies used in this study were anti-mouse

Cy7 (GL-1),

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CD11c-FITC (N418) or CD11c-BV480 (HL3), CD80-PerCPCy5.5 (16-10A1), CD86-PE and IAd-AlexaFluor647 (39-10-8) (Biolegend, San Diego, CA, USA),

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CD40-PE (5C3) (BD Biosciences Pharmingen, San Jose, CA, USA) antibodies. For BLT-1 detection, cells were cells were incubated in fixation/permeabilization buffer

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(eBioscience, Carlsbad, CA, USA) with rabbit anti-BLT1 receptor polyclonal antibody

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(Cayman Chemical, Ann Arbor, MI, USA) for 1 h, washed, followed by 1 h incubation with FITC Donkey anti-rabbit IgG (poly4064, Biolegend) in the same buffer. Cells incubated with the secondary antibody only were used as control for background

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fluorescence. For Treg detection, splenocytes were stained with the Mouse Regulatory T Cell Staining Kit #1 (Affymetrix eBiosciences, San Diego, CA, USA) according to the manufacturer’s instructions. Cells were analyzed using a FACS Canto II flow cytometer (BD Biosciences). Results were obtained from at least 20,000 cells and analyzed using the FlowJo software (Tree Star, Ashland, USA). Doublets and dead cells were excluded. DCs were characterized as being CD11c+/MHCII+, and by the expression of

co-stimulatory molecules. Median fluorescence intensity (MFI) was determined on CD11c+ gated cells. T cell proliferation was determined by CD4+-gated cells with low expression of CFSE.

Detection of cytokines and LTB4

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Cytokines concentration was measured using OptEIA Set ELISA kits (BD Pharmingen) and LTB4 by the LTB4 EIA kit (Cayman Chemical, Ann Arbor, MI, USA)

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according to manufacturer’s instructions.

Statistical analyses

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The results are shown as mean values ± SEM. Statistical differences between were determined using one-way ANOVA, followed by the Newman-Keuls test or

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values were considered statistically significant.

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Student’s t test, using GraphPad Prism 5.0 software (San Diego, CA, USA). P < 0.05

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Results

The effects of exogenous LTB4 in DCs

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LTB4 acts on G protein-coupled receptors BLT-1 and BLT-2 (high and low affinity, respectively) which are both expressed in DCs [6]. To investigate if the

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expression of these receptors could be modulated by exogenous LTB4, we stimulated BM-DCs with LTB4 (10 nM) for 24 h and we found that BLT-1 protein expression was also found increased after the addition of LTB4, while no changes were observed when

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cells were treated with LPS (Fig. 1A). BLT-1 (Ltbr1) gene expression was also found increased by LTB4 whereas BLT-2 (Ltbr2) expression was not significantly affected (Fig. 1B). LTB4 was reported to induce the TLR adaptor molecule MyD88 expression and amplify its downstream activation in a BLT-1-dependent fashion in macrophages [2]. We observed that MyD88 expression was also increased in DCs after LTB4

stimulation (Fig. 1C). LPS (1 µg/mL) was used as a control stimulus and was also observed to increase both BLT-1 and Myd88 expression, but failed to induce endogenous LTB4 production. DCs stimulated for 24 h with 10 µg/mL of zymosan, a well-known LT inducer, were employed as a control (Fig. 1D). Because of the BLT-1 upregulation induced by LPS, we suspected that endogenous LTB4 could be produced

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and consumed by DCs at an earlier stage of LPS stimulation. We then assessed the presence of LTB4 in the culture’s supernatants at earlier time points (30 min, 1 h, 2 h, 4

h, and 8 h) but we did not find a significant increase of the mediator after LPS stimulus

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(data not shown).These results allowed us to exclude a potential effect of endogenousinduced LTB4 in our study and suggested that exogenous LTB4 could potentiate

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responses of TLR/IL-1R agonists in MyD88-dependent signaling pathway in DCs. To test this hypothesis, we examined the effect of LTB4 on LPS-induced cytokines

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production in DCs.

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Cells were treated with different doses of LTB4 (1, 10, or 100 nM) and cytokines were assessed for cell cultures’ supernatants after 24 h. Figure 2A show that LTB4

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alone, at the concentrations assayed, did not induce IL-6 or IL-12p40 but it significantly increased IL-10 production at the concentration of 10 nM. As expected, the addition of

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LPS (1µg/mL) greatly enhanced IL-6, IL-10, IL-12p40, and LTB4 (10 nM) was able to

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potentiate LPS-induced IL-10 and IL-12p40 production (Fig. 2B).

LTB4 on DCs’ antigen-presenting function Maturation of DCs is characterized by the upregulation of MHC II and co-

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stimulatory molecules’ expression and is induced by pro-inflammatory stimuli, such as TLR ligands. We then evaluated the effects of LTB4 treatment in the expression of these DC markers. After 24 h of LTB4 and/or LPS stimulation, DCs were characterized as CD11c+/MHCII+ cells. We found that LTB4 increased the expression of CD86 in DCs to similar levels of that induced by LPS. This effect was already maximal with 1 nM of

LTB4. However, the expression of CD40, CD80, and MHC II were not modulated by LTB4. The addition of 10 nM of LTB4 to LPS did not enhance any of the markers expression (Fig. 3). We next examined the consequences of LTB4 stimulation of DCs on their antigen-presenting function. For that, we used an in vitro antigen-specific lymphocyte

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proliferation assay where DCs were loaded with OVA (100 µg/mL) and stimulated with LTB4 (10 nM). After 24 h, cells were extensively washed to remove free OVA and

incubated with CFSE-labeled DO11.10 mice splenocytes for 72 h and antigen-induced

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T cell proliferation was assessed by flow cytometry. (CD4+/CFSElow). Figure 4A shows

that LTB4 stimulation on DCs significantly increased the antigen-specific lymphocytes

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proliferation in the co-cultures. Similarly to unstimulated DCs, in the absence of OVA, DCs stimulated with LTB4 did not induce T cell proliferation (data not shown). Next, we

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examined the expression of transcription factors in the co-cultures. Figure 4B shows

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that in the co-cultures of lymphocytes and DCs previously treated with LTB4 we found

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increased expression of Gata3 and Foxp3, transcription factors related to Th2 and Treg

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cell differentiation, respectively. The expression of Tbet or Rorγt (transcription factors for Th1 and Th17 cells, respectively) was not affected. These results suggested that

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DCs that had been previously stimulated with LTB4 had a tendency to induce Th2/Tregs lymphocytes priming. To confirm these findings, we further investigated the

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percentage of proliferating cells (CD4+/CFSElow) that were Foxp3+ in the co-cultures and we observed that LTB4-stimulated DCs induced higher number of Tregs (Fig. 4C). We also measured IL-13 in the supernatants of the co-cultures and we found that the

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cytokine levels were also increased when DCs had been stimulated with LTB4 (Fig. 4D). IL-4 was also measured but was not detected.

Discussion

In the first part of this work we confirmed that LTB4 induces Blt-1 expression in murine bone marrow derived DCs, corroborating previous findings [14].

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observed that DCs were able to respond to LTB4 stimulation, which exerted a positive loop on the high affinity BLT-1 receptor expression. We then stimulated DCs with LPS in order to assess endogenous LTB4 production in our model. LPS had already been

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shown to induce LTB4 in human alveolar macrophages and on BM-DCs that had been differentiated in the presence of GM-CSF, IL-4, and TNF-α [15, 16]. Surprisingly, LPS stimulation did not induce LTB4 production by our DCs. This may indicate that different

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protocols of BM-DC differentiation can affect the type of lipid mediators they will

produce as ours consisted of culturing murine BM cells with GM-CSF only. Jozefowski

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et al. [8] had also observed that in a similar setting as ours, LPS was not able to induce leukotriene release. In this study, they additionally discuss that a two-day treatment

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treatment, but no further tests with LPS.

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with IL-4 caused LT release suppression instead of stimulation upon zymosan

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We had previously compared BM cells differentiated by GM-CSF alone and

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GM-CSF+IL4 and we found that the addition of IL-4 to the differentiation stage of BMDCs induced a partial maturation of the cells after 7 days, meaning that the expression

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of co-stimulatory molecules and secretion of inflammatory cytokines was higher at basal conditions than when cells had been differentiated with GM-CSF exclusively

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(data unpublished). This was also observed by Labeur et al. [17] who described an intermediate maturation phenotype of BM-DCs cultured with GM-CSF+IL-4. More recently, Tu et al. [18] also reported that high levels of IL-4 promoted a mature DC

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phenotype from precursor cells. In our hands, the addition of IL-4 to the differentiation stage of BM-DCs, didn’t alter the yield of the CD11c+/MHCII+ population at the end of 6 or 7 days of cell culture, neither its antigen-presenting function so we kept our BM-DC differentiation protocol without additional recombinant IL-4. It is still to be determined the importance of IL-4 in the BM-DC generation regarding LT production.

Previous works from one of us have shown that LTB4 is a strong inducer of MyD88 expression, the intracellular adaptor molecule for toll-like receptors, in murine macrophages and thus potentiated LPS-induced cytokines production by macrophages [2, 19]. Here we showed for the first time that LTB4 also increased Myd88 expression in DCs. We also showed that 10 nM of LTB4 potentiated LPS-induced IL-10 and IL-

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12p40, but it didn’t affect IL-6 production by DCs. 1 µM of LTB4 combined with 200 ng/mL of LPS was also reported to induce IL-10 production, but on the contrary of our

finding, it seemed to decrease IL-12p40 release [8]. It is, however, noteworthy to

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highlight that 10 nM of LTB4 alone was also able to increase the production of the antiinflammatory IL-10 by DCs.

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We then investigated the effect of LTB4 on DCs’ antigen presenting function. During maturation process, besides being able to secrete pro- and anti-inflammatory

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factors, DCs upregulate the expression of MHC II and co-stimulatory molecules, which

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allow them to prime T cells, inducing adaptive immune responses. When combined

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with LPS, LTB4 did not induce further upregulation of these molecules and we assumed

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that LPS alone was already a robust maturation stimulus which probably affected the upregulation of those maturation markers to their maximum. On the other hand, we

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found that LTB4 alone was able to increase the expression of the co-stimulatory molecule CD86 but failed to upregulate CD40, CD80, and MHC II. LTB4 is well-known

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for its role in the stimulation of polymorphonuclear leukocytes [20-22] and was also implicated in the chemotaxis of DCs through the upregulation of CCR7 expression [6]. In our study, we found that LTB4 treatment also allowed DCs to induce higher antigen-

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specific CD4+ T cell proliferation, which confirmed LTB4 as maturation factor for DCs and points out that lipid mediators could have a relevant role in DCs antigen presentation to lymphocytes. Indeed, we and others have previously shown that other lipid mediators, platelet-activating factor and prostaglandin E2, were also involved in the maturation of murine BM-DCs with consequences on their T cell priming activity [23, 24].

Previous studies with DCs differentiated from BLT1-deficient mice led to impaired Th1 and Th17 responses due to reduced production of IL-6, IL-12, TNF-α, indicating an important role of this pathway in the triggering of these inflammatory responses [10, 25]. However, when we examined the phenotype of the total cells in the co-cultures of LTB4-stimulated DCs, we didn’t observe any alterations in Tbet or RorγT

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gene expression, Th1 and Th17 markers respectively. On the other hand, we found higher expression of Gata3 and Foxp3 genes which are respectively Th2 and Treg

phenotype markers. In addition to this, the Th2 response signature cytokine IL-13 was

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found in the supernatants of the co-cultures. The finding that LTB4-stimulated DCs leads to preferential activation of Th2 immune response, as shown here, could be of

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clinical relevance in allergic disease. There are several studies in experimental asthma that show that the development of the Th2 response is dependent on activation of

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LTB4/BLT-1 axis [6, 7, 9, 26, 27]. This axis may be also relevant in diabetes mellitus

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type 1 in which high systemic levels of LTB4 were shown in mice that could be involved

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in the development of the co-morbidities associated with the disease [19].

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Besides inducing a Th2-biased T cell proliferation, we showed that LTB4-treated DCs also promoted regulatory T cells differentiation/proliferation. This increase in

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FoxP3+ Tregs in the co-cultures is likely related to the CD86 upregulation observed after DCs were stimulated with LTB4. It is known that co-stimulation is required for

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Tregs homeostasis, especially in the control of autoimmune diseases [28, 29]. Thus, it is possible that DCs exposed to the antigen in an inflammatory environment rich in LTB4, by increasing the expression of different co-stimulatory molecules and inducing

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distinct cytokines production, would modulate T cells response to antigen by creating a more tolerogenic environment. Further studies on inhibition of LTB4 synthesis or blocking its receptors in disease models could open new therapeutic possibilities. In conclusion, this work presents novel insights on the effect of LTB4 in adaptive immunity through DC modulation disclosing not only relevance in homeostasis but also

in pathological states when LTB4 levels would be dysregulated. This knowledge may also be a relevant tool when modulation of T cells by DCs antigen-presentation is required, such as in cellular therapy.

Conflict of Interests - The authors declare that there is no conflict of interest regarding

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the publication of this article.

Acknowledgements - The authors are grateful to Marlise Montes for technical assistance. This work was supported by Fundação de Amparo a Pesquisa do Estado de

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São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e

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Tecnológico (CNPq).

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29.

Figure Caption

Figure 1 – Leukotriene B4 increases BLT-1 receptor and MyD88 expression in DCs. Murine DCs (106 cells/ mL – final volume of 2 mL) were stimulated with LTB4 (10 nM), LPS (1 µg/mL), or zymosan (10 µg/mL), a potent LTB4 inducer used as a positive

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control. After 24 h, LTB4 BLT-1receptor was assessed by Flow cytometry (A), the gene expression of LTB4 receptors (BLT-1 and BLT-2) (B) and of MyD88 (C) was determined

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by qPCR, and LTB4 production (D) was assessed by ELISA. qPCR results are shown as fold change of the untreated control group. (n=5). *P<0.05 vs. control group;

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#P<0.05 vs. LTB4-treated DCs.

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Figure 2 – LTB4 potentiates LPS-induced cytokines in DCs. Murine DCs (106

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cells/mL) were incubated with LTB4 (1, 10, 100 nM), LPS (1 µg/mL) or LTB4 + LPS (10

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nM and 1 µg/mL, respectively). Cytokine production was assessed by ELISA in the

LPS-stimulated DCs.

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supernatants after 24 h of stimulation (n=5). *P<0.05 vs. control group; #P<0.05 vs.

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Figure 3 – The effect of LTB4 on DC maturation markers. Murine DCs (106 cells) incubated with LTB4 (1, 10, and 100 nM), LPS (1 µg/mL) or LTB4+LPS (10 nM and 1

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µg/mL, respectively) for 24 h had the expression of CD86, CD80, CD40, and MHCII determined by flow cytometry and expressed in MFI as % of non-stimulated DCs. (n=4-

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6). *P<0.05 vs. control group.

Figure

4

–

LTB4

potentiates

DC-induced

antigen-specific

lymphocyte

proliferation. Murine DCs (106 cells) were stimulated with LTB4 (10 ng/mL) and pulsed with OVA (100 µg/mL) for 24 h. Then, DCs were extensively washed and co-cultured with CFSE-labeled DO11.10 splenocytes for 3 days. CD4+ T cell proliferation

(CD4+/CFSElow cells) and FoxP3+ Treg percentage of cells were assessed by flow cytometry (A and C), and the expression of transcription factors in the proliferating cells was assessed by qPCR (B). Supernatants of the co-cultures were tested for cytokines

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by ELISA (D). (n=9). *P<0.05 vs. control group.

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