The p110δ subunit of class IA PI3K modulates signaling in innate immune cells. We previously demonstrated that mice harboring a kinase-dead p110δ subunit (p110δKD) develop spontaneous colitis. Macrophages contributed to the Th1/Th17 cytokine bias in p110δKD mice through increased IL-12 and IL-23 expression. In this study, we show that the enteric microbiota is required for colitis development in germfree p110δKD mice. Colonic tissue and macrophages from p110δKD mice produce significantly less IL-10 compared with wild-type mice. p110δKD APCs cocultured with naive CD4+ Ag-specific T cells also produce significantly less IL-10 and induce more IFN-γ– and IL-17A–producing CD4+ T cells compared with wild-type APCs. Illustrating the importance of APC–T cell interactions in colitis pathogenesis in vivo, Rag1−/−/p110δKD mice develop mild colonic inflammation and produced more colonic IL-12p40 compared with Rag1−/− mice. However, CD4+CD45RBhigh/low T cell Rag1−/−/p110δKD recipient mice develop severe colitis with increased percentages of IFN-γ– and IL-17A–producing lamina propria CD3+CD4+ T cells compared with Rag1−/− recipient mice. Intestinal tissue samples from patients with Crohn’s disease reveal significantly lower expression of PIK3CD compared with intestinal samples from non–inflammatory bowel disease control subjects (p < 0.05). PIK3CD expression inversely correlates with the ratio of IL12B:IL10 expression. In conclusion, the PI3K subunit p110δ controls homeostatic APC–T cell interactions by altering the balance between IL-10 and IL-12/23. Defects in p110δ expression and/or function may underlie the pathogenesis of human inflammatory bowel disease and lead to new therapeutic strategies.
Genetic variants that confer susceptibility to the human inflammatory bowel diseases (IBD) Crohn's disease (CD) and ulcerative colitis highlight the importance of innate immune interactions with the enteric microbiota in both initiating and controlling inflammation (1). Commensal and pathogenic microorganisms are recognized through conserved molecular microbial patterns by pattern-recognition receptors, of which TLRs form integral components (2). Although mechanisms by which the host distinguishes commensal from pathogenic bacteria are not well defined, under normal conditions TLR signaling initiated by the enteric microbiota is protective (3). PI3Ks have emerged as important regulators of TLR signaling (4, 5). Class IA PI3Ks have five different regulatory subunits and three p110 catalytic subunits: p110α and p110β are expressed ubiquitously in tissues, whereas p110δ is enriched in leukocytes (6). Agents that activate macrophages to produce IL-12p40, the common subunit of the proximal inflammatory cytokines IL-12 and IL-23, also activate class IA PI3K (7). Activation of PI3K in turn blocks the expression of IL-12p40 mRNA (Il12b) (7). Although inflammatory responses are essential for eradicating pathogenic microbes, excessive/prolonged activation of innate immunity is harmful to the host. PI3K-mediated negative feedback of IL-12p40 is important to prevent excessive innate immune responses.
The clearest role for PI3K in chronic inflammation is described in a mouse harboring a kinase-dead p110δ catalytic subunit of PI3K (p110δD910A/D910A kinase dead; hereafter referred to as p110δKD) (8). These mice demonstrate B and T cell defects, including defective Ag receptor signaling and impaired humoral responses. Notably, the occurrence of spontaneous colitis was demonstrated in PI3K p110δKD mice (9). Expression of IL-12p40, Th1, and Th17 cytokines was described in the intestinal and systemic immune compartments. Consistent with a homeostatic role for p110δ in the intestine, wild-type (WT) mice raised in a germfree (GF) environment markedly upregulated colonic p110δ (Pik3cd) expression when the enteric microbiota were introduced, but colitis-prone Il10−/− mice raised in the same conditions did not (9).
Given the role of the PI3K p110δ subunit in innate immune processes fundamental to the pathogenesis of IBD, host-enteric microbiota and APC–T cell interactions in p110δKD mice were further characterized. We describe a requirement for the enteric microbiota to drive intestinal inflammation in p110δKD mice. Microbial-innate immune interactions, through p110δ, maintain homeostasis through regulation of both protective (IL-10) and inflammatory (IL-12p40) cytokines. Furthermore, p110δ orchestrates innate regulation of adaptive immune responses. Importantly, in human CD, decreased intestinal PIK3CD gene expression is demonstrated that inversely correlates with intestinal IL12B:IL10 ratios. Thus, p110δ appears to be a central homeostatic switch in the intestine, governing the critical balance between microbiota-induced IL-12/23 and IL-10, shaping the subsequent T cell response. Counter to prevailing paradigms in which p110δ inhibition is a strategy in inflammatory diseases (10, 11), induction of p110δ could be a potential therapeutic approach in human IBD.
Materials and Methods
All mice were maintained on a C57BL/6 background in conventional or GF housing. PI3K p110δD910A/D910A (p110δKD) mice were previously obtained from B. Vanhaesebroeck (Queen Mary University of London, London, U.K.). GF p110δKD mice were Caesarian derived, as previously described (12), and were maintained according to standard techniques in the University of North Carolina National Gnotobiotic Resource Center. OT-II [C57BL/6-Tg(TcraTcrb)425Cbn/J] male mice were provided by J. Ting (University of North Carolina, Chapel Hill, NC). All animal experiments were in compliance with protocols approved by the International Animal Care and Use Committee of the University of North Carolina at Chapel Hill.
LPS from Escherichia coli and Pam3CSK4 were purchased from Invivogen (San Diego, CA). Zymosan A from Saccharomyces cerevisiae was purchased from Sigma-Aldrich (St. Louis, MO). Inhibitors IC87114, rapamycin, and SB-216763 were purchased from Selleck Chemicals (Houston, TX). Cecal bacterial lysates (CBL) from C57BL/6 mice were prepared, as described previously (13). The peptide corresponding to residues 323–339 of OVA was purchased from AnaSpec (Fremont, CA).
Colonic tissue explant culture
Colonic tissue explant cultures were performed, as described previously (14).
Slides were prepared for H&E staining, and a pathologist (L.B.B.) blinded to the study groups performed histological analysis using established criteria for p110δKD mice (9). In T cell adoptive transfer studies, the following scoring system was used: tissue changes were categorized into inflammatory and epithelial changes and graded for severity (0 = normal, 1 = mild, 2 = moderate, 3 = marked, and 4 = severe); the sum of the two grades comprised the histopathology score. For inflammation, a score of 0 (normal) signified rare small lymphoplasmacytic aggregates confined to the lamina propria; scores of 1 (mild) and 2 (moderate) represented increasing numbers of multifocal inflammatory aggregates that were predominantly confined to the lamina propria, with occasional submucosal infiltration; a score of 3 (marked) was assigned if inflammatory infiltrates frequently extended into the submucosa and muscular layers; a score of 4 (severe) was designated if transmural inflammation was common. Epithelial changes, characterized by hypertrophy, were scored (0–4) with increasing severity and prevalence of the observed change.
Bone marrow–derived macrophages (BMDMs) were cultured, as described previously (15). Splenocytes were isolated, as described (16), and further separated into CD11c+ and CD11c−/Cd11b+ cells by MACS with anti-CD11c and anti-CD11b microbeads (Miltenyi Biotec, Auburn, CA). Lamina propria mononuclear cells (LPMCs) were isolated from mouse colons, as described previously (17). LPMCs were further separated into CD11b+ and CD11b− cells by MACS with anti-CD11b microbeads (Miltenyi Biotec).
Cell culture experiments
BMDMs or splenocytes were cultured at 1 × 106/ml in the presence of LPS (1 ng/ml), zymosan A (10 μg/ml), Pam3CSK4 (5 ng/ml), or PBS, and supernatants were harvested after 4 or 24 h (BMDMs or splenocytes, respectively). Inhibitors IC87114 (0.1 or 1 μM), rapamycin (1 or 10 μM), SB-216763 (1 or 10 μM), or DMSO were added 1 h prior to stimulation with LPS or zymosan A. CD11b+ LPMCs were treated with IC87114 (10 μM) for 30 min prior to exposure to heat-killed E. coli (HKEC; multiplicity of infection [MOI] = 100) for 3 h. Total RNA was assessed for Il12b and Il10 expression by quantitative PCR.
Western blot analysis
BMDMs were cultured at 1 × 106/ml with LPS (1 μg/ml), or cecal tissue from GF to conventionalized (CNV) mice was collected. Cell or tissue lysates were collected in radioimmunoprecipitation assay buffer with protease and phosphatase inhibitors. Equal amounts of protein were loaded and run on a 10% SDS-PAGE gel. Blots were probed for phosphorylated glycogen synthase kinase-3β (GSK-3β) (Ser9) and total GSK-3β with Abs from Cell Signaling Technology (Danvers, MA) and phosphorylated CREB (Ser133) (EMD Millipore, Billerica, MA) and total CREB (Santa Cruz Biotechnology, Dallas, TX). Equal protein loading was confirmed by probing blots with anti-β-actin (mAbcam 8226) from Abcam (Cambridge, MA).
Quantitative RT-PCR was performed on total RNA, as described (14). Murine primer sequences will be provided upon request. The following human primer sequences were used: PIK3CD, forward, 5′-GCGCCGGGACGATAAGGAGTC-3′, and reverse, 5′-GCTGCCCACAGGGGTCTACCT-3′; IL10, forward, 5′-GCCTAACATGCTTCGAGATC-3′, and reverse, 5′-TGATGTGTGGGTCTTGGTTC-3′; IL12B, forward, 5′-GCTCTTGCCCTGGACCTGAACGC-3′, and reverse, 5′-CGTAGAATTGGATTGGTATCCGG-3′; GAPDH, forward, 5′-GGTGAAGGTCGGAGTCAACGGA-3′, and reverse, 5′-GAGGGATCTCGCTCCTGGAAGA-3′.
IL-12p40 and IL-10 (BD Biosciences, San Jose, CA) and IFN-β concentrations (R&D Systems, Minneapolis, MN) were determined by sandwich ELISA, according to manufacturer’s instructions.
APC–CD4+ T cell coculture
Splenic APCs from WT or p110δKD mice were isolated by negative selection using CD90.2 microbeads (Miltenyi Biotec). Splenic APCs were incubated overnight with CBL (50 ng/ml), and, after washing several times to remove extracellular Ag, APCs were cocultured with negatively selected CD4+ T cells (CD8α/B220/MHC II microbeads; Miltenyi Biotec) from WT or Il10−/− mice at a 3:2 ratio (APC:T cell) for 72 h. For Ag-specific studies, APCs were incubated overnight with LPS (10 ng/ml) and OVA peptide (323–339, 5 μM) (13). After washing to remove extracellular Ag, APCs were cocultured with negatively selected CD4+ T cells (CD8α/B220/MHC II microbeads; Miltenyi Biotec) from mice expressing a transgenic TCR that recognizes OVA epitope residues 323–339 (OT-II mice) at a 3:2 ratio (APC:T cell) for 72 h. CD4+ T cells were analyzed for intracellular cytokine expression (IFN-γ and IL-17A) by flow cytometry.
CD4+ T cells were stimulated for 4 h with PMA (100 ng/ml) and ionomycin (1 μg/ml) in the presence of GolgiStop (BD Biosciences). Cells were then washed and stained with allophycocyanin-conjugated anti-CD3 (clone 17A2; eBioscience, San Diego, CA). After fixing and permeabilizing the cells with BD Cytofix/Cytoperm (BD Biosciences), staining for intracellular PE-conjugated anti–IFN-γ (clone XMG1.2; eBioscience) and FITC-conjugated anti–IL-17A (clone eBio17B7; eBioscience) was performed. Flow cytometry samples were run on a CyAn ADP Analyzer (Beckman Coulter, Brea, CA) and analyzed using Summit v4.3 (Beckman Coulter).
CD4+CD45RBhigh/low T cell–adoptive transfer colitis
T cell–mediated colitis was induced in Rag1−/− and Rag1−/−/p110δKD (RKO/δKD) mice at 8 wk of age, as described previously (18). CD4+ T cells were isolated by negative selection (CD8α/B220/MHC II microbeads; Miltenyi Biotec) and stained with FITC-conjugated anti-CD4 (clone GK1.5; eBioscience) and PE-conjugated anti-CD45RB (clone 16A; BD Pharmingen, San Jose, CA). CD4+ T cells were sorted into CD45RBhigh and CD45RBlow populations using a MoFlo XDP Cell Sorter (Beckman Coulter). Mice were i.p. injected with 4 × 105 CD4+CD45RBhigh cells admixed with 2 × 105 CD4+CD45RBlow cells, as described (18). Clinical scores were assigned, as described (19).
Human intestinal samples
Intestinal samples were obtained from surgical resections from CD patients and subjects requiring surgical intervention for noninflammatory conditions (e.g., colon cancer). In CD patients, when available, paired inflamed and noninflamed intestinal segments, as determined by gross appearance by the processing pathologist, were obtained for analysis. The University of North Carolina Institutional Review Board approved collection of de-identified samples, and written informed consent was obtained from all patients.
Statistical significance for data subsets was assessed by the two-tailed Student t test. The p values <0.05 were considered to be significant. All data are expressed as mean ± SEM.
Presence of the enteric microbiota is necessary for the development of colitis in p110δKD mice
To determine whether the microbiota is necessary for the development of colitis, p110δKD mice were derived GF. GF p110δKD mice up to 30 wk of age did not develop histological colitis (Fig. 1A, Supplemental Fig. 1A). Interestingly, GF p110δKD mice produced significantly less colonic IL-10 compared with GF WT mice (Fig. 1B, left). GF p110δKD and WT mice were then transitioned to CNV housing, and colonic inflammation was assessed at days 7 and 14 after transfer. Compared with GF to CNV WT mice, colons from GF to CNV p110δKD mice demonstrated increased colitis scores (Fig. 1A, Supplemental Fig. 1A). However, GF to CNV p110δKD mice gained weight similarly to GF to CNV WT mice (Supplemental Fig. 1B). Furthermore, colonic explants from day 7 and 14 GF to CNV p110δKD mice produced significantly less IL-10 (Fig. 1B) compared with GF to CNV WT mice. At day 14, GF to CNV p110δKD mice produced significantly elevated IL-12p40 (Fig. 1C) compared with WT GF to CNV mice. IL-10 is important for the maintenance of intestinal homeostasis in part through inhibition of IL-12p40 (20). The ratio of colonic IL-12p40 to IL-10 protein production therefore reflects the overall balance of intestinal pro- and anti-inflammatory cytokines. Indeed, colons from days 7 and 14 GF to CNV p110δKD mice demonstrated significantly higher ratios of IL-12p40:IL-10 production (Fig. 1D) compared with GF to CNV WT mice.
Given the recent report of coregulation of IL-10 and IFN-β by p110δ in dendritic cells (21), we measured IFN-β levels in colonic explant tissue cultures from GF to CNV WT and p110δKD mice. Interestingly, IFN-β production was enhanced in colonic explants from day 7 GF to CNV p110δKD mice compared with WT mice (Fig. 1E), in contrast to decreased levels of IL-10 production in p110δKD mice. IFN-β production was increased in LPS-stimulated p110δKD compared with WT BMDMs (Fig. 1F). Additionally, LPS-stimulated Il10−/− BMDMs produced more IFN-β compared with WT BMDMs (Fig. 1F), consistent with previously reported regulation of IFN-β by IL-10 (21). To assess the direct contribution of IFN-β production from colonic macrophages, CD11b+ LPMCs, comprising mostly macrophages, were isolated from p110δKD mice and stimulated with HKEC. Similar to BMDMs, HKEC-stimulated p110δKD CD11b+ LPMCs produced significantly more IFN-β compared with WT CD11b+ LPMCs (Fig. 1G), suggesting that p110δ differentially regulates IL-10 and IFN-β in macrophages.
PI3K p110δ regulates macrophage production of IL-10 in response to pathogen-associated molecular patterns
WT and p110δKD BMDMs were exposed to TLR agonists (LPS [TLR4], 5 ng/ml; Pam3CSK4 [TLR2/1], 5 ng/ml; zymosan A [TLR2/6], 5 μg/ml), and cytokine production was measured. BMDMs from p110δKD mice produced less IL-10 in response to all TLR agonists tested compared with WT BMDMs (Fig. 2A). Additionally, p110δKD BMDMs exposed to TLR agonists produced significantly more IL-12p40 compared with WT BMDMs (Fig. 2B), in agreement with our previously published data (9). Consequently, the ratio of IL-12p40:IL-10 production in TLR ligand–treated p110δKD BMDMs was consistently increased compared with WT BMDMs (Fig. 2C). LPS- or zymosan A–stimulated CD11b+ and CD11c+ splenocytes from p110δKD mice also produced less IL-10 and more IL-12p40 than WT splenic cells (Supplemental Fig. 2A–D).
To further validate these findings, LPS-stimulated WT BMDMs were treated with p110 isoform–specific chemical inhibitors. LPS-activated WT BMDMs demonstrated a dose-dependent decrease in IL-10 production (Fig. 3A) and increase in IL-12p40 (Fig. 3B, left) with specific chemical inhibition of p110δ (IC87114). Interestingly, p110δ inhibition in WT BMDMs markedly enhanced IL-12p40 production above levels produced by p110δKD BMDMs. It is possible that there is compensation by other PI3K isoforms in p110δKD BMDMs but not in chemically inhibited WT BMDMs. Indeed, we have previously shown that phosphorylation of Akt in LPS-stimulated p110δKD BMDMs is not completely abrogated (9). PI3K p110α and p110β inhibition (PIK-90 and TGX-221, respectively) did not alter LPS-stimulated IL-10 production (Supplemental Fig. 2E, 2F) or IL-12p40 (Supplemental Fig. 2G, 2H) in WT BMDMs, in agreement with reported results in dendritic cells (21). As a control, p110δ-specific inhibition of LPS-activated p110δKD BMDMs did not alter IL-10 or IL-12p40 expression (Fig. 3A, 3B). However, in p110δKD BMDMs, p110β inhibition decreased IL-10 (Supplemental Fig. 2F) production, and p110α- or p110β-specific inhibition modestly enhanced LPS-induced IL-12p40 expression (Supplemental Fig. 2G, 2H), suggesting that, in the absence of p110δ function, other isoforms may have modest effects on IL-10/IL-12p40 regulation.
Chemical inhibition of p110δ in Il10−/− BMDMs led to a dose-dependent increase in IL-12p40 production (Fig. 3C), suggesting that p110δ-mediated decreases in IL-12p40 are in part independent of the inhibitory actions of IL-10. Relevant to mucosal innate inflammatory responses, WT CD11b+ colonic LPMC treated with HKEC demonstrated diminished Il10 (Fig. 3D) and enhanced Il12b (Fig. 3E) expression in the presence of the p110δ-specific inhibitor. As a control, expression of neither cytokine was altered in CD11b+ colonic LPMC from p110δKD mice treated with the p110δ-specific inhibitor.
Mammalian target of rapamycin and GSK-3β act downstream of p110δ in macrophages to regulate cytokine production
PI3Ks modulate multiple downstream signaling pathways, of which mammalian target of rapamycin (mTOR) and GSK-3β have been previously shown to regulate cytokine secretion in macrophages (22). PI3K p110δKD BMDMs treated with LPS demonstrated impaired phosphorylation of GSK-3β and CREB (Fig. 4A, 4B, respectively), but not p70 S6 kinase (downstream of mTOR; data not shown). Furthermore, colonic expression of p-GSK-3β was attenuated in GF to CNV p110δKD mice compared with GF to CNV WT mice (Fig. 4C). BMDMs from WT and p110δKD mice were exposed to mTOR or GSK-3β inhibitors (rapamycin or SB-216763, respectively) prior to LPS stimulation. Rapamycin decreased IL-10 (Fig. 5A, 5C) and increased IL-12p40 (Fig. 5B, 5D) protein and mRNA expression in WT and p110δKD TLR-stimulated BMDMs. These same trends were observed in WT and p110δKD CD11b+ and CD11c+ splenocytes (Supplemental Fig. 3A–D). Inhibition of GSK-3β in p110δKD BMDMs and splenocytes increased IL-10 protein (Fig. 5E, Supplemental Fig. 3E, 3F) and mRNA expression (Fig. 5G), whereas p110δKD BMDMs and splenocytes decreased IL-12p40 protein (Fig. 5F, Supplemental Fig. 3G, 3H) and mRNA (Fig. 5H) expression. Hence, mTOR and GSK-3β are downstream of p110δ and are relevant for regulation of IL-10 and IL-12p40.
APC p110δ regulates T cell cytokine production
To begin to determine whether resident APCs regulate intestinal T cell phenotype and function, T cell cytokines and lineage markers were measured in p110δKD colons. Colonic Tbx21 and Rorc transcripts (Fig. 6A, 6B), the hallmark transcription factors of Th1 (23) and Th17 cells (24), respectively, were increased in GF to CNV p110δKD mice at days 7 and 14 posttransition compared with matched GF to CNV WT mice. Likewise, increased Ifng and Il17a transcripts (Fig. 6C, 6D) were detected in cecal tissue from days 7 and 14 GF to CNV p110δKD compared with matched GF to CNV WT mice.
Consequently, we next investigated whether T cell–dependent IL-12p40 and IL-10 expression was altered in p110δKD APCs. Splenic CD4+ T cells from WT mice were cultured with either WT or p110δKD splenic APCs pulsed with CBL. CBL-pulsed p110δKD APCs cultured with naive WT CD4+ T cells produced decreased levels of IL-10 (Fig. 6E) and increased levels of IL-12p40 (Fig. 6F) compared with WT APCs. WT and p110δKD APCs cultured with Il10−/− CD4+ T cells demonstrate that IL-10 expression is largely derived from APCs (Fig. 6E). As expected, CBL-pulsed p110δKD APCs also produced significantly less IL-10 (Fig. 6E) and more IL-12p40 (Fig. 6F) compared with WT APCs in the absence of CD4+ T cells.
Next, to study Ag-specific APC–T cell interactions, splenic CD4+ T cells from OVA-specific transgenic TCR mice (OT-II mice) were cocultured with OVA-pulsed and LPS-activated WT and p110δKD APCs. OVA-loaded p110δKD APCs induced significantly more IFN-γ–producing (Fig. 6H, 6I) and IL-17A–producing (Fig. 6H, 6J) CD4+ T cells compared with OVA-loaded WT APCs (Fig. 6G, 6I, 6J). However, T cell proliferation was induced to a similar extent by both WT and p110δKD APCs (Supplemental Fig. 4A–C). These data suggest that cytokine production by p110δKD APCs directs differentiation of Ag-specific Th1 and Th17 CD4+ T cells.
These results suggest a model in which defective p110δ, through regulation of IL-10 and IL-12p40, leads to inflammatory effector T cell development. To test this model in vivo, we generated Rag1−/−/p110δKD mice (RKO/δKD). Interestingly, colitis is present but attenuated in the absence of an adaptive immune system (Fig. 7A, 7B). Colonic explant cultures from RKO/δKD mice produced significantly decreased IL-10 (Fig. 7C) and increased IL-12p40 (Fig. 7D) compared with colonic tissue explant cultures from Rag1−/− mice.
It was previously reported that p110δKD CD4+ T cells adoptively transferred into Rag1−/− recipients induce colitis owing to impaired T regulatory cell function (25). To study how p110δ inactivation in nonlymphocyte populations affects T cell differentiation, admixed WT CD4+CD45RBhigh and CD4+CD45RBlow T cells were adoptively transferred into Rag1−/− and RKO/δKD mice (CD45RB recipient mice) and monitored for colitis development. Adoptive transfer of T cells demonstrated reconstitution, with localization of CD3+ cells to the colons of recipient mice (Supplemental Fig. 4D). Total body weight of recipient mice was recorded until the experiment was terminated at day 24 due to severe clinical manifestations in the CD45RB recipient RKO/δKD mice. There was no difference in weight loss between CD45RB recipient Rag1−/− and RKO/δKD mice (data not shown). Clinical colitis activity scores (Fig. 8A) and quantitative colonic histologic analysis (Fig. 8B, 8C) from CD45RB recipient RKO/δKD mice were increased compared with the respective recipient Rag1−/− mice. Colonic IL-10 production was significantly lower (Fig. 8D) and IL-12p40 production higher (Fig. 8E) in CD45RB recipient RKO/δKD mice compared with CD45RB recipient Rag1−/− mice. Consequently, ratios of colonic IL-12p40:IL-10 production from CD45RB recipient RKO/δKD mice were significantly higher (Fig. 8F) than ratios from recipient Rag1−/− mice. Furthermore, a greater percentage of IFN-γ–producing (Fig. 8G), but not IL-17A–producing (Fig. 8H), CD4+ T cells were isolated from mesenteric lymph nodes (MLNs) of CD45RB recipient RKO/δKD mice compared with recipient Rag1−/− mice. Finally, a greater percentage of lamina propria CD3+CD4+ T cells from recipient RKO/δKD mice produced IFN-γ (Fig. 8I) and IL-17A (Fig. 8J; each data point represents three pooled samples), compared with recipient Rag1−/− mice.
Intestinal PIK3CD expression correlates with IL12B:IL10 ratios from patients with CD
Expression of p110δ (PIK3CD), IL-12p40 (IL12B), and IL-10 (IL10) mRNA was determined in human intestinal tissue from control subjects without intestinal inflammation and patients with CD. Significantly higher levels of PIK3CD mRNA were detected in noninflamed intestinal samples from control subjects compared with tissue from patients with CD (Fig. 9A). Paired macroscopically inflamed and noninflamed intestinal resections from the same subject were obtained from 14 CD patients. There was lower expression of PIK3CD in inflamed intestinal tissues compared with noninflamed tissues obtained from the same patient (Fig. 9B). Furthermore, ratios of IL12B:IL10 expression from individual CD patients demonstrated a strong and statistically significant inverse correlation with PIK3CD expression (Fig. 9C).
We previously described the development of spontaneously occurring Th1- and Th17-mediated colitis in p110δKD (9). In the present series of experiments, we further elucidate intestinal host-microbial and APC–T cell interactions mediated by p110δ. Colitis in p110δKD mice is dependent on host responses to the enteric microbiota, as has been described in other murine colitis models (26). In the absence of the enteric microbiota, p110δKD mice did not develop intestinal inflammation, whereas, after reconstitution with commensal enteric microbiota, colons from p110δKD mice demonstrated histological inflammation, impaired IL-10, and increased IL-12p40 production (Fig. 1). Consequently, altered IL-10 and IL-12p40 production by p110δKD APCs in response to microbial products and cognate interactions with T cells orchestrates pathogenic adaptive immune responses contributing to intestinal inflammation.
Class IA PI3Ks regulate macrophage and dendritic cell responses to bacteria (5). Taken together, our results and those of others (21) elucidate a model in which p110δ is an intracellular integrator of environmental signals that is involved in the restoration of inflammatory responses to homeostasis, mediated in part by IL-10. Regulation of IL-10 expression involves both PI3K-dependent and independent pathways. Moreover, IL-10 signaling in macrophages has been shown to activate the PI3K pathway (27). Indeed, we have shown that colonic p110δ expression is attenuated in colitis-prone Il10−/− mice, suggesting that IL-10 regulation of IL-12p40 occurs in part via induction of p110δ (9). Our current and previous findings (9) demonstrate that bacterial products and IL-10 induce p110δ gene expression in macrophages and suggest that transcription regulation of p110δ can determine functional immunologic consequences. Most studies have focused on posttranslational regulation of p110δ (21, 28, 29) as the sole biologic determinant of function. Thus, transcriptional regulation of p110δ expression in macrophages may have unexpected biological significance and needs to be further explored.
Aksoy et al. (21) recently demonstrated that p110δ signaling in dendritic cells dampens responses to LPS by sequestering TLR4 signaling components and facilitating the switch from Toll–IL-1R domain-containing adaptor protein/MyD88-dependent inflammatory cytokine production (IL-12, IL-6, TNF-α) to Toll–IL-1R domain–containing adaptor inducing IFN-β (TRIF)–related adaptor molecule/TRIF-dependent anti-inflammatory cytokine production (IFN-β, IL-10). It is possible that LPS-induced p110δ signaling in macrophages also facilitates the switch to TRIF-related adaptor molecule–TRIF signaling, leading to the enhanced production of IL-10 and IFN-β. Indeed, this agrees with our finding that p110δKD macrophages produce less LPS-induced IL-10. However, we have previously shown that CD11b+ LPMCs from Trif−/− mice produce higher levels of basal and bacterially stimulated IL-10 compared with WT mice (30, 31). Conversely, BMDMs from Trif−/− mice produce less LPS-induced IL-10 compared with WT mice (32). These findings suggest that the TRIF pathway negatively regulates IL-10 production uniquely in intestinal macrophages. Furthermore, CD11b+ LPMCs produce high levels of IL-10 in GF conditions (30), suggesting that microbial signals are not necessary for driving constitutive expression of IL-10. However, TLR signaling, perhaps through the recognition of endogenous ligands, remains vital for IL-10 production in intestinal macrophages, as HKEC-stimulated MyD88−/− CD11b+ LPMCs do not produce detectable levels of IL-10 (30). Additionally, our data suggest that IFN-β and IL-10 demonstrate differential regulation in CD11b+ LPMCs, in contrast to the recent study demonstrating convergent regulation in LPS-stimulated BMDCs (21). Indeed, Kaiser et al. (33) demonstrated cell type–specific differences in IL-10 and IFN-β production in response to LPS: BMDMs and splenic macrophages did not make detectable amounts of IFN-β but made significant amounts of IL-10 in response to LPS, whereas BMDCs produced both IFN-β and IL-10, suggesting that macrophages use distinct pathways to regulate IFN-β and IL-10. Thus, further studies are necessary to elucidate specific intestinal macrophage signaling pathways required for IL-10 production.
In macrophages, mTOR and GSK-3β are central regulators of IL-12p40 and IL-10 downstream of PI3K. Bacterial products induce MyD88-dependent PI3K activation, leading to phosphorylation of its downstream effector molecule Akt. Akt inactivates tuberous sclerosis complex, a negative regulator of mTOR (34). Both Akt- and PI3K-dependent mTOR activation modulate IL-12p40 and IL-10 production by suppressing GSK-3β activity (22, 35). GSK-3β constitutively represses IL-10 by blocking CREB binding to and activation of the Il10 promoter (36). PI3K- and mTOR-mediated inhibition of GSK-3β thus releases IL-10 from suppression by GSK-3β (36). Interestingly, mTOR activation targets were not altered in LPS-stimulated p110δKD BMDCs compared with WT BMDCs, suggesting that compensatory activation pathways sustain mTOR signaling in p110δKD BMDCs (21). Indeed, mTOR is activated through many pathways, including cellular energy sensing and Wnt signaling (37). Additionally, the balance of IL-10 and IL-12p40 production, as well as other cytokines, is regulated by MAPK signaling downstream of TLR signaling (38). We previously reported that TLR-activated p110δKD macrophages demonstrate impaired Akt phosphorylation and enhanced phosphorylation of p38 and JNK MAPK, but not ERK1/2, compared with WT macrophages (9). In this study, we show impaired phosphorylation of the downstream mediators GSK-3β and CREB in TLR-stimulated p110δKD macrophages and in colons from GF to CNV p110δKD mice. Furthermore, other cytokines such as TNF-α are affected by p110δ, which regulates tubule fusion in TNF-α–containing vesicles bound for secretion (39). Indeed, we have previously shown dysregulation of IL-12p70, IL-23, and NO in TLR-activated p110δKD macrophages (9). Thus, p110δ may regulate cytokine secretion in multiple ways. In this study, we showed that inhibition of GSK-3β rescues LPS-induced IL-10 production in p110δKD BMDMs (Fig. 5E, 5G). Our results therefore suggest that GSK-3β activity may be a therapeutic target in IBDs to induce IL-10 production. Indeed, GSK-3β inhibition has previously been shown to ameliorate colitis in mice (40).
PI3K p110δKD B and T lymphocytes demonstrate impaired proliferative signaling through the B cell and TCRs (8). PI3K p110δKD mice demonstrate impaired intrinsic T regulatory cell function, and p110δKD CD4+CD45RBlow cells cotransferred with colitogenic WT CD4+CD45RBhigh cells did not protect Rag1−/− mice from T cell–mediated colitis (25). To determine how APC p110δ influences T cell subset differentiation and colitis development, APC–CD4+ T cell coculture experiments were performed (Fig. 6). CD4+ T cells induced greater production of IL-12p40 by APCs, whereas APCs from p110δKD mice induced more Ag-specific IFN-γ– and IL-17A–producing T cells. Because only WT CD4+ T cells were used in coculture with APCs from both WT and p110δKD mice, T cell phenotype can be attributed to the defect in p110δKD APCs. As an in vivo correlate, Rag1−/− and RKO/δKD mice were reconstituted with admixed WT CD4+CD45RBhigh and CD45RBlow T cells (Fig. 8). Compared with respective recipient Rag1−/− mice, RKO/δKD recipient mice demonstrated significantly increased clinical and histology scores. More IFN-γ–producing T cells were isolated from MLNs and colonic lamina propria of RKO/δKD recipient mice compared with recipient Rag1−/− mice. IL-17–producing CD4+ T cells are rarely found in MLNs and other secondary lymphoid tissues but are found in abundant quantities at mucosal surfaces (41). Indeed, RKO/δKD recipient mice contained significantly higher percentages of IL-17A–producing T cells in the colonic lamina propria compared with the respective Rag1−/− recipient mice. We previously showed that bacterially stimulated p110δKD macrophages produce significantly more IL-23, a cytokine necessary for the differentiation and maintenance of Th17 cells (9).
Although the development of pathogenic CD4+ T cells in CD45RB recipient mice was explored, we did not study T regulatory cell differentiation and function. During GF to CNV transition, colons from p110δKD mice demonstrated significantly increased transcription of Foxp3, correlating with increased inflammation, compared with colons from WT mice (data not shown). However, this finding does not rule out functional defects in p110δKD T regulatory cells. It is entirely possible, and in fact likely, akin to human IBD pathogenesis, that innate and adaptive immune defects interact to drive colitis.
Interestingly, in the absence of T cells, RKO/δKD mice developed mild histopathologic colonic inflammation. The development of mild colonic inflammation in RKO/δKD mice could be explained by the presence of non–hepaticus Helicobacter species in our mouse colony (data not shown). The ability of H. hepaticus to induce innate immune-driven colonic inflammation in the absence of adaptive immune cells has been well described (42, 43).
Recently, p110δ inhibition has been targeted for the treatment of chronic rejection of tissue transplants, systemic lupus erythematosus, and certain lymphoid cell malignancies (10, 11, 44). Although preliminary clinical results are promising, this study highlights a potentially untoward consequence of p110δ inhibition—enhanced intestinal and innate inflammatory processes initiated by APCs.
We thank Dr. Bart Vanhaesebroeck for providing p110δKD mice.
This work was supported by National Institutes of Health Grants RO1 DK054452 (to S.E.P.) and F30 DK089692 (to E.C.S.) and University of North Carolina Center for Gastrointestinal Biology and Diseases Grant P30 DK34987 (Histology Core, Gnotobiotic Core, and Immunotechnologies Core).
The online version of this article contains supplemental material.
Abbreviations used in this article:
bone marrow–derived macrophage
cecal bacterial lysate
glycogen synthase kinase 3β
heat-killed Escherichia coli
inflammatory bowel disease
lamina propria mononuclear cell
mesenteric lymph node
multiplicity of infection
mammalian target of rapamycin
Toll–IL-1R domain–containing adaptor inducing IFN-β
The authors have no financial conflicts of interest.