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. 2020 Jan 15:8:2050312120901547.
doi: 10.1177/2050312120901547. eCollection 2020.

Activation of IL-10+ B cells: A novel immunomodulatory mechanism for therapeutic bacterial suspensions

Alberto Salazar  1   2 , Jane E Nieto  2 , Henry Velazquez-Soto  2 , Maria C Jiménez-Martínez  1   2
Affiliations

Activation of IL-10+ B cells: A novel immunomodulatory mechanism for therapeutic bacterial suspensions

Alberto Salazar et al. SAGE Open Med. .

Abstract

Objectives: Bacterial components are used to improve immune responses in patients with respiratory infections. Pharmacological formulations of bacterial components include a mixture of bacterial antigens, some of which are complete inactivated bacteria, that is, named bacterial suspensions; while others are fragments of bacteria, which are presented as bacterial lysates. Although bacterial lysates have been broadly used as immune-stimulators, the biological support for the therapeutic effectiveness of bacterial suspension has not yet been studied. Thus, the aim of our study was to investigate the immunological activity induced by bacterial suspension.

Methods: This work was an exploratory translational study. Peripheral blood mononuclear cells were obtained from healthy donors and cultured in time-dose dependent assays with a commercial bacterial suspension. Flow cytometry was used for phenotypic analysis and for determining soluble cytokines in culture supernatants.

Results: We observed that bacterial suspension activates B cells in a dose-dependent manner. Peripheral blood mononuclear cells were able to secrete IL-6 and IL-10 after 24 h of bacterial suspension stimulation. TLR2 expression was observed mainly on CD19+ CD38Lo B cells after 72 h of culture; remarkably, most of the TLR2+ CD19+ cells were also IL-10+.

Conclusion: Our findings suggest that bacterial suspension induces the activation of B cell subsets as well as the secretion of IL-6 and IL-10. Expression of TLR2 on CD19+ cells could act as an activation loop of IL-10+ B regulatory cells. The clinical implications of these findings are discussed at the end of this article.

Keywords: B cells; Bregs; IL-10; IL-6; bacterial suspension; immunomodulation.

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Conflict of interest statement

Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Percentage of CD69 expressing CD3+ and CD19+ cells after BS-14 stimuli. PBMCs were stimulated with 36.3 ng/mL of BS-14, and after 48 h of stimulation, then cells were harvested and stained with fluorescence-conjugated antibodies to CD3, CD19, and CD69 in a triple-immunofluorescence assay as described in the material and methods. Representative histograms of CD3+ and CD19+ gated cells are shown from five different healthy individuals and performed in triplicate.
Figure 2.
Figure 2.
Increased CD69 frequency on CD19+ CD38+ cells after BS-14 stimulation. CD19+ cells were gated according to CD38 expression and separated into three cell subsets: CD38Lo, CD38Med, and CD38Hi. Histograms from CD19+ CD38+ cells subpopulation are shown. The x-axis denotes the frequency of CD69 on the gated cells. PBMCs were stimulated with BS-14 at 36.3 ng/mL, and evaluated after 48 h of stimulation. Con A mitogen was used as a positive stimulation control. Representative FACS plots and histograms from five different healthy individuals are shown and were performed in triplicate.
Figure 3.
Figure 3.
Increased TLR2 frequency on CD19+ CD38+ cells after 72 h of BS-14 stimulation. Percentage of TLR2+ cells in (a) CD19+ CD38Lo B cells, and in (b) CD19+ CD38Med B cells. Significant differences are indicated (*). n = 5 different healthy individuals were evaluated, and all assays were performed in triplicate.
Figure 4.
Figure 4.
TLR2+ cells are the main source of IL-10. After 72 h of stimulation with BS-14 at 36.3 ng/mL, TLR2 expression was assessed on CD19+ cells by flow cytometry. Intracellular IL-10 was evaluated in TLR2– and TLR2+ B cells. The histograms show CD19+ TLR2-IL-10+ (upper right) and CD19+ TLR2+ IL-10+ (low right), demonstrating that TLR2+ B cells are the primary source of IL-10. Representative FACS plots and histograms from five different healthy individuals, which were performed in triplicate.
Figure 5.
Figure 5.
The hypothetic sequence of events after stimulation of mononuclear cells with BS. BS-14 induces activation of B cells in a dose and time-dependent manner. B cell activation developed inside a microenvironment enriched with IL-6 and IL-10, which increased the expression of TLR2 on the B cells. A possible role for TLR2 on CD19+ cells is as a contributor in an activation loop of CD19+ CD38+ IL-10+ Breg subsets.
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