Elsevier

Experimental Cell Research

Volume 327, Issue 1, 10 September 2014, Pages 78-90
Experimental Cell Research

Research Article
PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment

https://doi.org/10.1016/j.yexcr.2014.05.016Get rights and content

Highlights

  • First genetic evidence that PTEN controls LPS/TLR4 signaling in B lymphocytes.

  • Evidence that PTEN regulates LPS induced lymphoproliferation in vivo.

  • PI-3 kinase inhibitors block LPS induced lymphoproliferation in vivo.

Abstract

Pattern recognition receptors (PRRs), e.g. toll receptors (TLRs) that bind ligands within the microbiome have been implicated in the pathogenesis of cancer. LPS is a ligand for two TLR family members, TLR4 and RP105 which mediate LPS signaling in B cell proliferation and migration. Although LPS/TLR/RP105 signaling is well-studied; our understanding of the underlying molecular mechanisms controlling these PRR signaling pathways remains incomplete. Previous studies have demonstrated a role for PTEN/PI-3K signaling in B cell selection and survival, however a role for PTEN/PI-3K in TLR4/RP105/LPS signaling in the B cell compartment has not been reported. Herein, we crossed a CD19cre and PTENfl/fl mouse to generate a conditional PTEN knockout mouse in the CD19+ B cell compartment. These mice were further crossed with an IL-14α transgenic mouse to study the combined effect of PTEN deletion, PI-3K inhibition and expression of IL-14α (a cytokine originally identified as a B cell growth factor) in CD19+ B cell lymphoproliferation and response to LPS stimulation. Targeted deletion of PTEN and directed expression of IL-14α in the CD19+ B cell compartment (IL-14+PTEN-/-) lead to marked splenomegaly and altered spleen morphology at baseline due to expansion of marginal zone B cells, a phenotype that was exaggerated by treatment with the B cell mitogen and TLR4/RP105 ligand, LPS. Moreover, LPS stimulation of CD19+ cells isolated from these mice display increased proliferation, augmented AKT and NFκB activation as well as increased expression of c-myc and cyclinD1. Interestingly, treatment of LPS treated IL-14+PTEN-/- mice with a pan PI-3K inhibitor, SF1126, reduced splenomegaly, cell proliferation, c-myc and cyclin D1 expression in the CD19+ B cell compartment and normalized the splenic histopathologic architecture. These findings provide the direct evidence that PTEN and PI-3K inhibitors control TLR4/RP105/LPS signaling in the CD19+ B cell compartment and that pan PI-3 kinase inhibitors reverse the lymphoproliferative phenotype in vivo.

Introduction

B cells are central elements in the adaptive immune response to pathogens. Antigen (Ag) recognition through the BCR and CD19 drives B cell activation and differentiation. B cells also display elements of innate immunity; they express several proteins of the PRR/TLR family including the LPS receptors, TLR4 and RP105 (CD180) [1], [2], [3] through which these cells recognize and respond to pathogen-associated ligands [3], [4]. TLR stimulation of B cells enhances their Ag presentation capacity and promotes cytokine secretion, cell proliferation, and differentiation into Ab secreting cells. TLR4 and RP105, are two of the most extensively studied TLRs, which recognizes gram negative bacterial LPS as its prototype agonist, in the presence of MD-2 and this recognition is facilitated by CD14 and LPS-Binding Protein (LBP) [2], [5]. TLR4 signaling utilizes both “MyD88-dependent” and “MyD88-independent” signaling pathways which require receptor dimerization, adapter recruitment, and the activation of specific kinases and transcription factors and result in inflammatory gene expression. RP105 is selectively expressed in mature CD19+ B cells, signals separate from MyD88, and uses CD19 as a coreceptor to signal through lyn, Vav, PI-3K, AKT, IKKα and BAFF-R to activate LPS dependent B cell proliferation in mantle zone (MZ) B cells [1], [2]. The activation of either of these pathways allows NF-κB to diffuse into the cell nucleus and activate transcription and consequent induction of inflammatory cytokines. In addition to the expression of inflammatory cytokines, NF-κB also promotes cell growth and differentiation through transcriptional regulation of c-myc and cyclin D1 [6], [7]. The role of TLR4 signaling in enhancing B lymphocyte trafficking into lymph nodes, B cell proliferation, polarization, migration and directionality [8], [9] is well-known.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a lipid phosphatase that dephosphorylates PI(3,4,5)P3 to PI(4,5)P2, is known to antagonize phosphoinositide 3-kinase (PI-3K) [10], [11]. The balance between PTEN and PI-3K determines PI(3,4,5)P3 levels and opposing effects on growth and cell survival [12]. PI(3,4,5)P3 is thought to mediate these effects by inducing phosphorylation/activation of the PDK1 and AKT kinases, which enhances the PIP3-PI-3K/AKT survival pathway [13]. The role of PTEN/phosphoinositide 3-kinase (PI-3K) in mediating TLR4 signaling has been studied in endothelial cells, macrophages and liver [14], [15], [16], [17], [18], [19], [20]. Li et al. has suggested the connection between MyD88 and PI-3K signaling pathways and showed that MyD88 TIR domain blocked LPS induced AKT activation in endothelial cells [14]. On the same note, work from other labs, reported the role of PI-3K in TLR4-mediated activation of NF-κB and COX-2, as well as IL-6, MCP-1, IFN-inducible protein 10, IL-12, and IL-10 [14], [21]. Recently, Luyendyk et al. [22] used Pik3r1-/- mice and PTEN-/- mice to study the role of PI-3K in TLR4 signaling and concluded that PI-3K plays negative role in TLR4 signaling in macrophages. In agreement with these results, Cao et al. has reported that PTEN supports TLR4 signaling in murine peritoneal macrophages [19]. Recently, Kamo et al. has provided a novel regulatory link between the PTEN mediated AKT/-β catenin/Foxo1 and TLR4 pathway and provide a mechanism for β-catenin-mediated immunomodulation in IR-stressed livers [20].

PTEN/PI-3K is a central signal transduction axis controlling normal B cell homeostasis and influencing normal B cell functional responses including the development of B cell subsets, antigen presentation, immunoglobulin isotype switch, germinal center responses and maintenance of B cell anergy [23], [24], [25], [26], [27], [28]. Recent studies reported the augmented activation of PI-3K signaling in diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) [29], [30]. In contrast, studies conducted by Anzelon et al. suggest that loss of PTEN alone in the B cell compartment does not result in transformation of B cells [28]. The deletion of PTEN and SHIP in the CD19+ compartment resulted in the formation of malignant lymphomas mostly of the MZ phenotype [31]. The IL-14α-transgenic mice is an excellent model for human Sjogren׳s disease, an autoimmune disease associated with mature B cell infiltration of the submandibular and parotid glands, hypergammaglobulinemia, pulmonary interstitial lung disease and, as with the CD19cre driven PTEN/SHIP-/- model, the development of mature B cell lymphomas in the mantle zone (MZ) compartment[32]. The IL14α transgenic mice were reported by Shen et al. to develop CD5+, CD19+, SIgM+, monoclonal immunoglobulin gene rearrangements consistent with mature B cell lymphomas at age of 14–18 months, also a mantle cell phenotype. [32]. Although IL14α has been linked to human lymphoma and autoimmune disease, almost nothing is known about IL14α signaling in B cells. We hypothesized that combined deletion of PTEN and expression of IL-14α in CD19+ B cell compartment will augment B cell proliferation in mature CD19+ B cell compartment and lead to the development of a distinct B cell lymphoma. In the present study, we used a CD19Cre x PTENfl/fl cross in combination with IL-14 transgenesis to evaluate the role of IL-14, PTEN and PI-3K inhibitor therapeutics in LPS-induced lymphoproliferation in the CD19+ B cell compartment. Herein, we provide experimental evidence that the targeted deletion of PTEN combined with the directed expression of IL-14α in LPS stimulated CD19+ B cells leads to the activation of AKT, splenomegaly and highly altered spleen morphology with no distinct red and white pulp. Furthermore, CD19+ cells isolated from IL-14+; PTEN-/- mice show increased LPS induced AKT activation, which leads to increased levels of cyclinD1 and c-myc and augmented NF-κB activation. These findings establish a central role of PTEN and PI-3K in controlling LPS signaling in CD19+ B cells and suggest that PI-3K inhibitors may have therapeutic activity in the control of this phenotype where the microbiome and inflammation impact mature B cell-mediated lymphoproliferative disease and autoimmunity.

Section snippets

Mice and lipopolysaccharide (LPS) treatment

IL-14α, PTENflox/flox (PTENfl/fl) and CD19-Cre mice were obtained from Dr. Ambrus and the Jackson Laboratory (Bar Harbor, ME), respectively. All procedures involving animals were approved by the University of California San Diego Animal Care Committee, which serves to ensure that all federal guidelines concerning animal experimentation are met. The PTEN floxed homozygotes were generated in Balb/c FVB mice [33], and extensively backcrossed into the C57BL/J genetic background (>100 backcrosses).

Generation of PTEN null IL-14α+ mouse model for the study of CD19+ B-cell biology

In order to generate a mouse model for the study of the role of PTEN in lymphoproliferation as well as in the regulation of LPS signaling in B cells, we generated a CD19 directed PTEN deficient mouse model. CD19cre x PTENfl/fl mice were crossed with IL-14α transgenic mice, which resulted in IL-14+/PTEN-/- genotype in the B cell compartment (Supplementary Fig. S1A). The genotypes used in this study WT, IL-14+, PTEN-/-, and IL-14+PTEN-/- are represented in Fig. 1A. Using a CD19Cre transgenic

Discussion

The role of LPS signaling in B cell proliferation, polarization and migration is well studied [8], [9]. However, molecular mechanisms controlling LPS signaling in B cells are unclear. In the present study, we established a central role of PTEN and PI-3K in regulating LPS signaling in the CD19+ B cell compartment.

IL-14 is a cytokine that was identified and cloned from a Burkitt lymphoma cell line [43] and shown to enhance B cell proliferation, especially of germinal center B cells and surface Ig

Conflict of interest statement

D. Durden discloses financial conflict of interest related to the development of SF1126. This aspect has been reviewed by the UCSD committee on conflict of interest.

Acknowledgment

This work was supported by NIH Grants CA94233 and HL091365 to Donald L. Durden.

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