PI3Kδ hyper-activation promotes development of B cells that exacerbate Streptococcus pneumoniae infection in an antibody-independent manner - PubMed (original) (raw)

doi: 10.1038/s41467-018-05674-8.

Anita Chandra 1 2 3 4, Krishnendu Chakraborty 1 3, Rafeah Alam 1, Valentina Carbonaro 1, Jonathan Clark 5, Srividya Sriskantharajah 6, Glyn Bradley 7, Alex G Richter 8 9, Edward Banham-Hall 1 3 4, Menna R Clatworthy 10, Sergey Nejentsev 3, J Nicole Hamblin 6, Edith M Hessel 6, Alison M Condliffe 11, Klaus Okkenhaug 12 13

Affiliations

PI3Kδ hyper-activation promotes development of B cells that exacerbate Streptococcus pneumoniae infection in an antibody-independent manner

Anne-Katrien Stark et al. Nat Commun. 2018.

Abstract

Streptococcus pneumoniae is a major cause of pneumonia and a leading cause of death world-wide. Antibody-mediated immune responses can confer protection against repeated exposure to S. pneumoniae, yet vaccines offer only partial protection. Patients with Activated PI3Kδ Syndrome (APDS) are highly susceptible to S. pneumoniae. We generated a conditional knock-in mouse model of this disease and identify a CD19+B220- B cell subset that is induced by PI3Kδ signaling, resides in the lungs, and is correlated with increased susceptibility to S. pneumoniae during early phases of infection via an antibody-independent mechanism. We show that an inhaled PI3Kδ inhibitor improves survival rates following S. pneumoniae infection in wild-type mice and in mice with activated PI3Kδ. These results suggest that a subset of B cells in the lung can promote the severity of S. pneumoniae infection, representing a potential therapeutic target.

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Conflict of interest statement

A.C. received support for travel and attendance at conferences from Shire, SCL-Behring, Biotest and Grifols. S.S., G.B., E.B.H., J.N.H., and E.M.H. are employed by GSK. A.C., A.M.C., and S.N. received research funding from GSK. K.O. has received speaker’s or consultancy fees from Karus Pharmaceuticals and Gilead Sciences. The remaining authors declare no competing interests.

Figures

Fig. 1

Fig. 1

Prophylactic, but not therapeutic treatment with the inhaled PI3Kδ inhibitor nemiralisib mitigates disease severity following S. pneumoniae infection in wild-type mice. Wild-type mice were treated twice daily with the inhaled PI3Kδ inhibitor nemiralisib for the duration of the study: when treatment was started 24 h prior to infection with S. pneumoniae serotype 4, TIGR 4, survival rates were improved. When started 8 or 24 h post-infection, the treatment had no effect on survival outcome. (−24 h: data from five independent experiments combined n = 60; +8 h/+24 h: data from three independent experiments combined n = 36; data-points represent individual animals)

Fig. 2

Fig. 2

PI3Kδ hyper-activation leads to increased PIP3 and pAKT levels that can be reduced using a selective PI3Kδ inhibitor. a PIP3 levels in purified T cells from wild-type, p110δE1020K and p110δD910A mice, unstimulated or stimulated with anti-CD3 and anti-CD28 in the presence or absence of the selective PI3Kδ inhibitor, nemiralisib (mean ± SD; n = 2–3). b PIP3 levels in purified B cells from wild-type, p110δE1020K and p110δD910A mice unstimulated, or stimulated with anti-IgM in the presence or absence of nemiralisib (mean ± SD n = 2–6). c Western blots of purified T cells from wild-type, p110δE1020K and p110δD910A mice, unstimulated or stimulated with anti-CD3 and anti-CD28 in the presence or absence of nemiralisib. d Western blots of purified B cells from wild-type, p110δE1020K and p110δD910A mice, unstimulated or stimulated with anti-IgM in the presence or absence of nemiralisib. (Representative of two independent experiments; data-points represent individual animals)

Fig. 3

Fig. 3

Phenotype of B cells in p110δE1020K-GL mice. a B cell subsets in the spleen and bone marrow from wild-type, p110δE1020K-GL and p110δD910A mice were analyzed by flow cytometry and representative pseudocolor plots with the mean cell proportion are shown. b In the spleen, the number of B1, marginal zone (MZ) and T1 transitional cells B cells were increased in p110δE1020K-GL mice while follicular B cell numbers were normal. These populations were reduced in p110δD910A mice. Analysis of the bone marrow showed normal pro-B cell numbers in p110δE1020K-GL mice with a reduced number of pre-B cells, immature, transitional and mature B cells. Populations of cells are described as follows: Splenic B cells: Total B cells CD19+B220+, B1 cells CD19+B220+CD23-CD21-, Follicular B cells CD19+B220+CD23+CD21+, Marginal zone B cells CD19+B220+CD23-CD21+, Transitional T1 B cells B220+CD93+IgM+CD23-, Transitional T2 B cells B220+CD93+IgM+CD23+, Transitional T3 B cells B220+CD93+IgM−CD23−; Bone marrow B cells - Immature B cells CD19+B220loIgM+, Mature B cells CD19+B220hiIgM+, Pro-B cells B220+IgM−CD19+CD25−, Pre-B cells B220+IgMCD19+CD25+, Transitional B cells B220+IgD+. (Mean cell numbers are shown; combined data from two independent experiments, wild-type n = 10; p110δE1020K-GL_n_ = 12; p110δD910A_n_ = 7. Data-points represent individual animals)

Fig. 4

Fig. 4

PI3Kδ hyper-activation leads to increased susceptibility to S. pneumoniae infection. a Germline p110δE1020K-GL mice show accelerated disease development and significantly increased mortality compared to control mice in response S. pneumoniae infection, while kinase dead p110δD910A mice do not respond differently to wild-type mice. b–c Naïve PI3KδE1020K mice produce normal levels of anti-phosphorylcholine IgM and significantly higher levels of anti-PC IgG, while PI3KδD910A mice produce no natural antibody. d–f The p110δE1020K mutation was introduced conditionally into myeloid cells, T cells and B cells by crossing onto _Lyz2_cre, _Cd4_cre and _Mb1_cre lines respectively. The myeloid conditional mutation did not affect survival following S. pneumoniae infection in p110δE1020K-M mice (d), while T cell conditional p110δ hyper-activation led to improved survival in p110δE1020K-T mice (e). Introducing the p110δE1020K mutation specifically in B cells (p110δE1020K-B mice) replicated the increased susceptibility to S. pneumoniae seen in p110δE1020K-GL mice (f). g Transfer of p110δE1020K-B bone marrow into irradiated RAG2−/− recipients also conferred increased susceptibility to S. pneumoniae infection compared to recipients receiving wild-type bone marrow, and this phenotype was not fully rescued in a 50% BM chimera. (Data-points represent individual animals; data from 2 independent experiments combined. a n = 24; b, c mean antibody levels shown, WT n = 11, E1020K n = 8, D901A n = 9; d n = 22; e n = 20; f n = 20; g n = 20)

Fig. 5

Fig. 5

PI3KδE1020K-GL mice respond to Pneumovax and have normal antibody levels. a, b Pneumovax (T-independent pneumococcal polysaccharide vaccine, PPV) partially protects p110δE1020K-GL mice against infection (a), while p110δD910A are not protected (b). c p110δE1020K-GL mice produce normal levels of total IgG in response to Pneumovax immunization (anti-pneumococcal serotype 4 shown) while p110δD910A mice do not respond to vaccine. (Data-points represent individual animals; a results from three independent studies combined, n = 30; b results from two independent studies combined, n = 19; c results from two independent studies combined: mean antibody levels shown; WT n = 22; E1020K n = 8; D910A n = 6)

Fig. 6

Fig. 6

B cells could drive increased pathology in response to S. pneumoniae infection, independent of bacterial clearance. _Ighm_tm1 (µMT) mice show delayed disease progression and improved overall survival up to 30 days post S. pneumoniae infection, however 41% (7/17) surviving _Ighm_tm1 mice failed to clear bacteria from the lung tissue at 30 days post-infection compared to 100% clearance in surviving wild-type mice. (Data from 1 study, n = 24, mean CFU count shown, data-points represent individual animals)

Fig. 7

Fig. 7

PI3Kδ signaling regulates B cell specific IL-10 production in the lung following S. pneumoniae infection. a 24 h post S. pneumoniae infection, lung and spleen CFU counts were similar in wild-type, p110δE1020K-GL and p110δD910A mice. b 24 h post-infection, cytokine levels in the lung homogenate showed a trend towards increased TNFα, IL-6, and IL-1 in p110δE1020K mice. c 24 h prophylactic treatment with nemiralisib (nem) led to reduced levels of TNFα, IL-6, IL-1β, IFNγ, and IL-10 in the lungs of wild-type mice compared to vehicle control (veh) treated animals at 24 h post-infection. d Volcano plot (statistical significance against fold change) of the gene expression changes in response to nemiralisib treatment showed reduced levels of pro-inflammatory cytokines as well as IL-10 at 24 h post infection compared to vehicle control treatment. All genes analyzed are shown (gray dots) with the cytokines of interest labeled and colored; green for those with a negative fold change, red for positive fold change. e Analysis of immune cell subsets in _Il10_ITIB reporter mice at 24 h post-infection showed that the proportion of IL-10 producing B cells is significantly increased in p110δE1020K-GL mice and reduced in p110δD190A mice compared to wild-type mice, with similar trends in T cells and myeloid cells not reaching significance. (Mean values shown; data-points represent individual animals. a Results from two independent studies combined, n = 14; b representative data from three independent studies n = 8; c Combined data from four independent experiments n = 25; d data from 1 study, n = 6; e representative data from two independent studies n = 6)

Fig. 8

Fig. 8

p110δE1020K mice have an expanded population of IL-10 producing CD19+B220− B cells. a Naïve p110δE1020K mice show a significant increase in the proportion and number of B220−B cells in bone marrow and spleen. While this population is detectable in wild-type mice, it is absent from p110δD910A mice. Representative pseudocolor plots show mean cell proportions in naïve mice b S. pneumoniae (S.p) infection does not lead to significant expansion or recruitment of B220− B cells at 24 h post-infection in bone marrow, blood, spleen or lung tissue. Representative pseudocolor plots show mean cell proportions in naïve mice. c At 24 h post S. pneumoniae infection, IL-10 production is increased in p110δE1020K B cells from both lung and spleen, and reduced in p110δD910A B cells compared to wild-type mice. Furthermore, B220−B cells produce higher levels of IL-10 in response to S. pneumoniae infection compared to B220+ B cells in wild-type and p110δE1020K mice. Representative pseudocolor plots show mean cell proportions in S. pneumoniae infected mice (Mean cell counts are shown, data-points represent individual animals. a Combined data from two independent experiments, WT n = 10; E1020K n = 12; D910A n = 7, b representative data from two independent experiments, n = 6, c representative data from two independent experiments n = 6)

Fig. 9

Fig. 9

IL-10 producing B220−B cell subset is a novel B cell subset, expanded in PI3KδE1020K mice. In order to compare surface marker expression from B220− and B220+ IL-10 producing B cells, splenocytes from _Il10_ITIB mice (WT and p110δE1020K) were stimulated with LPS/PdBu/Ionomycin/Brefeldin A for 5 h and then stained for cell surface markers as shown. a After gating on IL-10 producing B cells, CD43++B220− cells were compared to CD43−B220+ cells. Histograms highlighting the differential surface marker expression as measured by median fluorescence intensity (MFI) between these populations are shown. b Comparison of surface marker MFI show that CD19+ B220− IL-10+ B cells express: CD43++CD5int/+ CD23−CD21−CD1dlo/int IgM+/-IgDlo/-PDL1−CD138− as opposed to conventional Bregs expressing: CD19hi B220hi IL-10+ CD43− CD5VarCD23−CD21++CD1dhi IgMhi IgDVar/lo PDL1+CD138int. (mean MFI ± SD is shown, representative data from two independent experiments, n = 5; data-points represent individual animals)

Fig. 10

Fig. 10

Nemiralisib treatment reduces the proportion of IL-10 producing B cells in mice and humans. a Prophylactic treatment with nemiralisib improved survival in p110δE1020K-GL mice and was associated with a significant reduction in the proportion of IL-10 producing B220− B cells in the lung at 24 h post-infection. (a Combined data from two independent studies, survival n = 24; lung tissue analysis n = 12, mean cell number shown, data-points represent individual animals) b Blood from patients with APDS (n = 6) and healthy controls (n = 6) was obtained and the B cell phenotype was determined by flow cytometry. Transitional B cells are identified as CD19+IgD+CD27−CD24+CD38+. Representative contour plots with outliers show the mean proportions of cells. c Freshly isolated PBMCs were unstimulated or stimulated with plate bound anti-CD3 and anti-IL-2 for 72 h in the presence or absence of nemiralisib (n = 2–3). The proportion of IL-10 producing cells among the total B cell and transitional B cell populations was determined by flow cytometry. Representative contour plots with outliers show mean proportions of IL-10 producing cells. (Mean cell proportions are shown, data-points represent individual patients; Combined data from two independent experiments)

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