Phosphoinositide 3-Kinase P110δ-Signaling Is Critical for Microbiota-Activated IL-10 Production by B Cells that Regulate Intestinal Inflammation

The phosphoinositide 3-kinase catalytic subunit p110δ (PI3Kδ) gene maps to a human inflammatory bowel diseases (IBD) susceptibility locus, and genetic deletion of PI3Kδ signaling causes spontaneous colitis in mice. However, little is known regarding the role of PI3Kδ on IL-10-producing B cells that help regulate mucosal inflammation in IBD. We investigated the role of PI3Kδ signaling in B cell production of IL-10, following stimulation by resident bacteria and B cell regulatory function against colitis. In vitro, B cells from PI3KδD910A/D910A mice or wild-type B cells treated with PI3K specific inhibitors secreted significantly less IL-10 with greater IL-12p40 following bacterial stimulation. These B cells failed to suppress inflammatory cytokines by co-cultured microbiota-activated macrophages or CD4+ T cells. In vivo, co-transferred wild-type B cells ameliorated T cell-mediated colitis, while PI3KδD910A/D910A B cells did not confer protection from mucosal inflammation. These results indicate that PI3Kδ-signaling mediates regulatory B cell immune differentiation when stimulated with resident microbiota or their components, and is critical for induction and regulatory function of IL-10-producing B cells in intestinal homeostasis and inflammation.


Preparation for Quantification of Fecal Lipocalin-2
Fecal samples (10-20 mg) were incubated overnight at 4 • C in PBS containing 0.1% Tween 20 (Fisher Scientific) and vortexed shortly to obtain homogenous fecal suspensions. The fecal suspensions were centrifuged for 10 min at 12,000 rpm at 4 • C. Clear supernatants were collected and stored at −20 • C.

Histological Colitis Score
Intestinal tissues were removed and fixed in 10% buffered formalin. Paraffin-embedded sections (5 µm) were prepared and stained with hematoxylin and eosin (H&E) by the Histology Core of the Center for Gastrointestinal Biology and Disease at UNC. The scoring of mucosal inflammation in cecum, proximal colon and distal colon was performed in a blinded fashion, with each region being graded from 0 to 4 as described previously [35]. The total histology scores represent the summation of all the scores (maximum score of 12).

Statistical Analysis
Statistical analysis was performed with Prism 8 software (GraphPad, San Diego, CA, USA). Significance between two groups was determined by unpaired Mann-Whitney test, while significance between more than 3 groups was determined by analysis of variance (ANOVA), followed by Dunn's multiple comparisons test. For all statistical comparisons, P values less than 0.05 were considered significant.

Inflammatory (IL-12p40) or Regulatory (IL-10) Pathways are Determined by PI3Kδ Signaling When Stimulated by Resident Bacteria
We first confirmed that PI3Kp110δ D910A/D910A mutant (PI3Kδ D910A ) mice in SPF conditions spontaneously develop colitis beginning at the age of 14 weeks or older based on serial fecal lipocalin-2 (f-Lcn) levels ( Figure 1a) [38]. Histology assessment indicated that 8 week old PI3Kδ D910A mice did not show any mucosal inflammation, while 18 week old PI3Kδ D910A mice developed colitis characterized by mucosal crypt hyperplasia, lamina propria (LP) infiltration by neutrophils and lymphocytes and increased intraepithelial lymphocytes (Figure 1b,c) [12,31,32]. As inflammation modifies mucosal immune status [39][40][41], 8-10 week old mice were predominantly used for in vitro experiments. The size of MLNs and spleens are smaller and the length of colons is shorter in 8-10 week old PI3Kδ D910A mice compared to WT mice ( Figure S1a). The cell numbers of PI3Kδ D910A mice were decreased in the spleen, MLN and colonic LP (cLP) with striking reduction of frequencies in B cells ( Figure S1b,c), which might be due to impaired cell proliferation, differentiation or survival in PI3Kδ D910A mice [31,42].
We next performed a pharmacologic blockade of PI3K signaling on WT cells during bacterial product stimulation. IL-10 secretion by CBL-stimulated WT cLP, MLN and spleen cells was significantly inhibited by PI3K-global inhibitors (LY294002 and Wortmannin), PI3Kδ-selective inhibitor (IC87114) and an inhibitor of Akt, a molecule downstream of the PI3K pathway ( Figure 1f and Figure S2) [44]. However, these inhibitors had no effect on IL-12p40 secretion. The IL-10/IL-12p40 ratio, which in part reflects the regulatory activity of immune cells, was significantly reduced by the PI3K/Akt inhibitors ( Figure 1f and Figure S2). As cell viability confirmed by the trypan blue exclusion test was not different between the groups (data not shown), the limited IL-10 production by the PI3K/Akt inhibitors was neither due to the cytotoxicity nor induction of apoptosis by the inhibitors. The lack of suppression of IL-12p40 further confirmed the viability of cells after inhibitor treatment. Together, PI3K-Akt signaling appears to skew the bacterial-stimulated immune phenotype toward regulatory (IL-10) rather than inflammatory (IL-12p40) responses. (data not shown), the limited IL-10 production by the PI3K/Akt inhibitors was neither due to the cytotoxicity nor induction of apoptosis by the inhibitors. The lack of suppression of IL-12p40 further confirmed the viability of cells after inhibitor treatment. Together, PI3K-Akt signaling appears to skew the bacterial-stimulated immune phenotype toward regulatory (IL-10) rather than inflammatory (IL-12p40) responses.
In vitro, B cells were an important source of bacterial-stimulated IL-10 production (Figure 2d), and the frequencies of IL-10-producing B cells in PI3Kδ D910A MLN cell cultures following bacterial stimulation were significantly lower compared to those in WT cell cultures (Figure 2d,e). Of note, IL-10-producing B cells produced more IL-10 in response to bacterial stimulation than did regulatory T cells both in vivo (Figure 2a,b) and in vitro (Figure 2f). To further analyze the association of the PI3Kδ pathway in intestinal IL-10-producing B cells, cLP B cells from WT mice were cultured with PI3K/AKT inhibitors in the presence or absence of bacterial stimulation. CBL promoted regulatory activity in B cells, indicated by an increased IL-10/IL-12p40 ratio, while blockade of PI3K-global or PI3Kδ-specific pathways significantly reversed this effect by selectively inhibiting secretion of IL-10 but not IL-12p40 (Figure 2g). These observations suggest that the PI3K signaling pathway is important for the IL-10-mediated regulatory function of bacteria-induced IL-10-producing B cells. In addition, we observed relatively lower fluorescence intensity of GFP (IL-10) in PI3Kδ D910A ; Il10 eGFP B cells compared to WT; Il10 eGFP B cells ( Figure S3), indicating functionally low activity of IL-10-producing PI3Kδ D910A B cells. To identify the mechanisms underlying the lower IL-10 secreting ability in PI3Kδ D910A ; Il10 eGFP B cells, we examined the surface markers in intestinal B cells. As we demonstrated recently, the phenotype of intestinal regulatory B cells in WT mice is IgM low IgD low CD23 low CD24 high [6]. In contrast, gut PI3Kδ D910A ; Il10 eGFP B cells show different expression patterns characterized by higher levels of IgM, IgD, and CD23, and lower expression of CD24 compared to WT B cells ( Figure S4), indicating potentially different responses to stimuli. Together, these results suggest that a reduced number and functional properties of IL-10-producing B cells may contribute to the pathogenesis of intestinal inflammation in PI3Kδ D910A mice.

Bacteria-Stimulated PI3Kδ D910A B Cells Do Not Suppress Pro-Inflammatory Cytokine Secretion by Macrophages
Intestinal macrophages are a major source of cytokines in IBD [1,3,4]. To investigate the anti-inflammatory function of regulatory B cells on bacterial-activated macrophages, splenic B cells from either PI3Kδ D910A or WT mice were co-cultured with the macrophage cell line J774, as a source of pro-inflammatory innate cytokines [1,3,4] (Figure 3a). PI3Kδ D910A B cells produced less IL-10 than did WT B cells when stimulated by bacteria components (Figure 3b), with further induction of IL-10 by WT, but not PI3Kδ D910A , B cells in co-culture with J744 macrophages. Surprisingly, macrophages alone did not secrete IL-10 in the absence of B cells. LPS-, CpG-and CBL-induced IL-12p40 and TNF-α production by macrophages was significantly suppressed by co-cultured WT but not PI3Kδ D910A B cells (Figure 3c,d). These results indicate that IL-10-producing B cells regulate macrophage responses to enteric bacterial ligands and that PI3Kδ signaling is required for B cell inhibition of macrophage activation by bacterial products. The percentages of GFP + T cells (CD45 + TCRβ + CD3 + ) and B cells (CD45 + B220 + CD19 + ) in MLN cells (CD45 + ) stimulated with CBL are shown in f. (g) 5 × 10 6 cLP B cells magnetically isolated from WT mice were cultured with or without CBL (10 µg/mL) in the absence or presence of PI3K/Akt inhibitors or vehicle control as described in Figure 1 legend in complete medium for 24 h. IL-10 and IL-12p40 levels in supernatants were measured by ELISA and IL-10/IL-12p40 ratios were calculated. Each symbol represents the result from individual mice, and bars indicate the mean in a-c, and e. Mean ± SEM are shown in f,g. Mann-Whitney unpaired two-tailed test was used for a-c, e, one-tailed for f. Dunn's multiple comparisons test following one-way ANOVA was used for g. *p < 0.05, **p < 0.01. ND indicates not detected. NS indicates not significant. The percentages of GFP + T cells (CD45 + TCRβ + CD3 + ) and B cells (CD45 + B220 + CD19 + ) in MLN cells (CD45 + ) stimulated with CBL are shown in f. (g) 5 × 10 6 cLP B cells magnetically isolated from WT mice were cultured with or without CBL (10 µg/mL) in the absence or presence of PI3K/Akt inhibitors or vehicle control as described in Figure 1 legend in complete medium for 24 h. IL-10 and IL-12p40 levels in supernatants were measured by ELISA and IL-10/IL-12p40 ratios were calculated. Each symbol represents the result from individual mice, and bars indicate the mean in a-c, and e. Mean ± SEM are shown in f,g. Mann-Whitney unpaired two-tailed test was used for a-c, e, one-tailed for f. Dunn's multiple comparisons test following one-way ANOVA was used for g. *p < 0.05, **p < 0.01. ND indicates not detected. NS indicates not significant.

Secreted IL-10 is the Dominant Way B Cells Regulate Pro-Inflammatory Cytokine Production in Macrophages in Response to Bacterial Products
We next sought to determine the mechanisms of WT B cells' anti-inflammatory effects. Although humoral factors, especially secreted IL-10, are a key mediator of regulatory B cell activity direct cell-tocell contact can also mediate B cells regulatory function [45,46]. Thus, we evaluated which factor is more predominantly involved in the bacteria-stimulated regulatory activity in B cells against inflammatory macrophages. To explore these possibilities, murine bone marrow-derived macrophages were cultured in the supernatants (excluding cells) from bacterial product-stimulated WT or PI3Kδ D910A B cells ( Figure  4a). As expected, IL-10 levels in PI3Kδ D910A B cell supernatants were lower than those in WT B cell supernatants (Figure 4b). Pro-inflammatory IL-12p40 and TNF-α produced by the macrophages were significantly decreased with the supernatants from WT B cells, but only partially suppressed by the supernatants from PI3Kδ D910A B cells (Figure 4c). Blockade of IL-10 signaling by anti-IL-10 receptor antibody negated the regulatory effect of the supernatant from WT B cells, indicating that the antiinflammatory effect was primarily mediated by IL-10 ( Figure 4d). These results suggest that incomplete suppression by the PI3Kδ D910A B cell supernatant is primarily due to lower IL-10 concentrations. Moreover, as secreted IL-10 also affects B cells [45], we blocked IL-10 receptor on B cells to observe autocrine effect of IL-10. Interestingly, IL-10R blockade significantly increased IL-12p40 secretion in a dose-dependent manner in CpG-stimulated B cells ( Figure S5). This finding indicates that low levels of IL-10 secretion from PI3Kδ D910A B cells influence not only other surrounding cells but also the B cell responsiveness to bacterial stimuli. PI3Kδ signaling appears to modulate B cell phenotype both directly (by regulating TLR signaling) and indirectly (through an IL-10 autocrine pathway).

Secreted IL-10 is the Dominant Way B Cells Regulate Pro-Inflammatory Cytokine Production in Macrophages in Response to Bacterial Products
We next sought to determine the mechanisms of WT B cells' anti-inflammatory effects. Although humoral factors, especially secreted IL-10, are a key mediator of regulatory B cell activity direct cell-to-cell contact can also mediate B cells regulatory function [45,46]. Thus, we evaluated which factor is more predominantly involved in the bacteria-stimulated regulatory activity in B cells against inflammatory macrophages. To explore these possibilities, murine bone marrow-derived macrophages were cultured in the supernatants (excluding cells) from bacterial product-stimulated WT or PI3Kδ D910A B cells (Figure 4a). As expected, IL-10 levels in PI3Kδ D910A B cell supernatants were lower than those in WT B cell supernatants (Figure 4b). Pro-inflammatory IL-12p40 and TNF-α produced by the macrophages were significantly decreased with the supernatants from WT B cells, but only partially suppressed by the supernatants from PI3Kδ D910A B cells (Figure 4c). Blockade of IL-10 signaling by anti-IL-10 receptor antibody negated the regulatory effect of the supernatant from WT B cells, indicating that the anti-inflammatory effect was primarily mediated by IL-10 ( Figure 4d). These results suggest that incomplete suppression by the PI3Kδ D910A B cell supernatant is primarily due to lower IL-10 concentrations. Moreover, as secreted IL-10 also affects B cells [45], we blocked IL-10 receptor on B cells to observe autocrine effect of IL-10. Interestingly, IL-10R blockade significantly increased IL-12p40 secretion in a dose-dependent manner in CpG-stimulated B cells ( Figure S5). This finding indicates that low levels of IL-10 secretion from PI3Kδ D910A B cells influence not only other surrounding cells but also the B cell responsiveness to bacterial stimuli. PI3Kδ signaling appears to modulate B cell phenotype both directly (by regulating TLR signaling) and indirectly (through an IL-10 autocrine pathway).

PI3Kδ-Signaling in B Cells Regulates Bacterial-Induced Pro-Inflammatory Cytokine Secretion by T Cells
As effector CD4 + T cells play a critical role in the pathogenesis of IBD [1,3,4], we explored the interactions between CD4 + T cells and B cells in intestinal inflammation. WT naïve CD4 + T cells plus Il10 −/− antigen presenting cells (APC) were co-cultured with WT, Il10 −/− , or PI3Kδ D910A B cells in the presence of CBL (Figure 5a). CBL activated naïve T cells to produce inflammatory IFN-γ and IL-17a in the presence of APC, while WT B cells [36], but not PI3Kδ D910A B cells, produced abundant IL-10 and decreased T cell inflammatory cytokines (Figure 5b). Furthermore, WT B cells significantly suppressed naïve CD4 + T cell proliferation, while either PI3Kδ D910A B cells or WT B cells plus PI3Kδ-inhibitors negated the suppressive effect of WT B cells on T cells (Figure 5c-e). These results indicate that B cells regulate CD4 + T cell proliferation and production of IFN-γ and IL-17 in response to bacteria and that PI3Kδ is required for these regulatory effects of B cells.

PI3Kδ-Signaling in B Cells Regulates Bacterial-Induced Pro-Inflammatory Cytokine Secretion by T Cells
As effector CD4 + T cells play a critical role in the pathogenesis of IBD [1,3,4], we explored the interactions between CD4 + T cells and B cells in intestinal inflammation. WT naïve CD4 + T cells plus Il10 −/− antigen presenting cells (APC) were co-cultured with WT, Il10 −/− , or PI3Kδ D910A B cells in the presence of CBL (Figure 5a). CBL activated naïve T cells to produce inflammatory IFN-γ and IL-17a in the presence of APC, while WT B cells [36], but not PI3Kδ D910A B cells, produced abundant IL-10 and decreased T cell inflammatory cytokines (Figure 5b). Furthermore, WT B cells significantly suppressed naïve CD4 + T cell proliferation, while either PI3Kδ D910A B cells or WT B cells plus PI3Kδ-inhibitors negated the suppressive effect of WT B cells on T cells (Figure 5c-e). These results indicate that B cells regulate CD4 + T cell proliferation and production of IFN-γ and IL-17 in response to bacteria and that PI3Kδ is required for these regulatory effects of B cells. (c-e) The 5 × 10 5 CFSE-labeled WT naïve CD4 + T cells were cultured with 1 × 10 6 B cells from WT or PI3Kδ D910A mice for 72 h with CBL (10 µg/mL). After culturing, single cells were stained and proliferation of naïve CD4 + T cells were assessed by flow cytometry. Representative histograms of live CD4 + T cells (Live/Dead neg CD45 + TCRβ + CD3 + CD4 + CD8 neg ) are shown in d. Percentage of proliferating cells was demonstrated in e. Bars indicate the mean. Dunn's multiple comparisons test following one-way ANOVA was used for b,e. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ND indicates not detected.

PI3Kδ-Signaling in B Cells is Required to Confer Protection Against T Cell-Mediated Colitis
Given the fact that the PI3Kδ pathway is involved in IL-10-mediated immune suppression by B cells in vitro, we investigated the involvement of B cell PI3Kδ in T cell-mediated colitis in vivo (Figure 6a). WT B cells decreased histologic inflammation in a CD4 + and B cell co-transfer colitis model with significantly less protection by either PI3Kδ D910A or Il10 −/− B cells (Figure 6b,c). These results were confirmed by decreased levels of fecal Lcn (Figure 6d). Expression of colonic inflammatory cytokine genes, Il12b, Ifng, Il17a and Tnfa were significantly ameliorated by WT B cells but less-so by PI3Kδ D910A and Il10 −/− B cells (c-e) The 5 × 10 5 CFSE-labeled WT naïve CD4 + T cells were cultured with 1 × 10 6 B cells from WT or PI3Kδ D910A mice for 72 h with CBL (10 µg/mL). After culturing, single cells were stained and proliferation of naïve CD4 + T cells were assessed by flow cytometry. Representative histograms of live CD4 + T cells (Live/Dead neg CD45 + TCRβ + CD3 + CD4 + CD8 neg ) are shown in d. Percentage of proliferating cells was demonstrated in e. Bars indicate the mean. Dunn's multiple comparisons test following one-way ANOVA was used for b,e. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ND indicates not detected.

PI3Kδ-Signaling in B Cells is Required to Confer Protection Against T Cell-Mediated Colitis
Given the fact that the PI3Kδ pathway is involved in IL-10-mediated immune suppression by B cells in vitro, we investigated the involvement of B cell PI3Kδ in T cell-mediated colitis in vivo (Figure 6a). WT B cells decreased histologic inflammation in a CD4 + and B cell co-transfer colitis model with significantly less protection by either PI3Kδ D910A or Il10 −/− B cells (Figure 6b,c). These results were confirmed by decreased levels of fecal Lcn (Figure 6d). Expression of colonic inflammatory cytokine genes, Il12b, Ifng, Il17a and Tnfa were significantly ameliorated by WT B cells but less-so by PI3Kδ D910A and Il10 −/− B cells (Figure 6e). Together, these in vivo results indicate that PI3Kδ signaling plays a pivotal role in IL-10-producing B cell protection in T cell-mediated colitis.
( Figure 6e). Together, these in vivo results indicate that PI3Kδ signaling plays a pivotal role in IL-10producing B cell protection in T cell-mediated colitis. (e) Expression of inflammatory cytokines in the distal colon was assessed by qPCR. Relative quantification was normalized to WT expression levels. Dunn's multiple comparisons test following one-way ANOVA was used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Discussion
The present study demonstrates that a lack of PI3Kδ signaling decreases bacteria-stimulated IL-10 secretion by B cells and impairs B cell function to inhibit inflammatory responses in macrophages and T cells. Adoptively transferred PI3Kδ D910A B cells were unable to attenuate T cell-mediated colitis. These findings indicate that PI3Kδ signaling is a key protective pathway for mucosal homeostasis and prevention of intestinal inflammation through bacterial-activated IL-10-producing B cells.
Dysfunction of the PI3Kδ pathway in mice leads to spontaneous colitis in the presence of resident bacteria and exacerbates mucosal inflammation in murine experimental colitis models [12,[30][31][32]. In humans, the PI3Kδ gene (PIK3CD) maps to the IBD7 susceptibility locus on chromosome 1p36 [28,29], while the PI3Kδ-inhibitor Idelalisib, a therapeutic agent for certain tumors, can cause colitis as a side effect [48]. Based on our present results showing that B cell PI3Kδ plays an anti-inflammatory role in the activation of macrophages and T cells and can attenuate T cell-mediated colitis, these clinical and basic observations might be in part due to PI3Kδ-mediated dysfunction of IL-10-producing regulatory B cells. Despite previous evidence showing dysfunction of multiple types of innate immune cells in PI3Kδ D910A mice [12,32], the present study showed that defective PI3Kδ signaling decreased the frequency and function of regulatory B cells in response to bacterial products to a greater extent than that of regulatory T cells or macrophages. Blockade of PI3Kδ signaling caused B cells to lose their ability to control bacterialactivated inflammatory cytokine production by macrophages or T cells. Finally, intact B cell-specific PI3Kδ signaling was sufficient to significantly ameliorate colitis in a T cell-transfer colitis model. These findings indicate that PI3Kδ signaling in B cells is a key determinant of the homeostatic phenotype of the mucosal immune system, and suggests that restoring or stimulating the PI3Kδ pathway in B cells might be a therapeutic target in IBD. (e) Expression of inflammatory cytokines in the distal colon was assessed by qPCR. Relative quantification was normalized to WT expression levels. Dunn's multiple comparisons test following one-way ANOVA was used. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Discussion
The present study demonstrates that a lack of PI3Kδ signaling decreases bacteria-stimulated IL-10 secretion by B cells and impairs B cell function to inhibit inflammatory responses in macrophages and T cells. Adoptively transferred PI3Kδ D910A B cells were unable to attenuate T cell-mediated colitis. These findings indicate that PI3Kδ signaling is a key protective pathway for mucosal homeostasis and prevention of intestinal inflammation through bacterial-activated IL-10-producing B cells.
Dysfunction of the PI3Kδ pathway in mice leads to spontaneous colitis in the presence of resident bacteria and exacerbates mucosal inflammation in murine experimental colitis models [12,[30][31][32]. In humans, the PI3Kδ gene (PIK3CD) maps to the IBD7 susceptibility locus on chromosome 1p36 [28,29], while the PI3Kδ-inhibitor Idelalisib, a therapeutic agent for certain tumors, can cause colitis as a side effect [48]. Based on our present results showing that B cell PI3Kδ plays an anti-inflammatory role in the activation of macrophages and T cells and can attenuate T cell-mediated colitis, these clinical and basic observations might be in part due to PI3Kδ-mediated dysfunction of IL-10-producing regulatory B cells. Despite previous evidence showing dysfunction of multiple types of innate immune cells in PI3Kδ D910A mice [12,32], the present study showed that defective PI3Kδ signaling decreased the frequency and function of regulatory B cells in response to bacterial products to a greater extent than that of regulatory T cells or macrophages. Blockade of PI3Kδ signaling caused B cells to lose their ability to control bacterial-activated inflammatory cytokine production by macrophages or T cells. Finally, intact B cell-specific PI3Kδ signaling was sufficient to significantly ameliorate colitis in a T cell-transfer colitis model. These findings indicate that PI3Kδ signaling in B cells is a key determinant of the homeostatic phenotype of the mucosal immune system, and suggests that restoring or stimulating the PI3Kδ pathway in B cells might be a therapeutic target in IBD.
Importantly, others have demonstrated contradictory roles for PI3Kδ in inflammatory diseases outside the intestine. Blockade of PI3Kδ attenuates inflammation in arthritis in K/BxN serum-transferred mice [49], IgE-or ovalbumin-related allergic models [50][51][52][53], a multiple sclerosis model [54], imiquimod-induced dermatitis [55], and experimental systemic lupus erythematosus in BXSB mice [56]. Inhibition of PI3Kδ signaling may predominantly suppress aggressive inflammatory immune cells over regulatory immune cells in non-IBD models, where resident intestinal bacteria are not the dominant activators of inflammation. Supporting this concept, pathogenic function of effector cells, such as neutrophils [49,51], CD8 + T cells [50,51], mast cells [50,51,53], Th2 cells [50][51][52], CD44 high CD62L low CD4 + T cells [54], Th17 cells [54][55][56], and autoreactive immunoglobulin-secreting B cells [56], were decreased by PI3Kδ signaling blockade in experimental inflammation outside the intestine. In contrast, aberrant activation of innate immune cells (macrophages, dendritic and antigen-presenting cells) and Th1/Th17 cells, in concert with decreased regulatory cells (regulatory T cells and B cells and intestinal dysbiosis), strongly contribute to the onset and progression of IBD and experimental intestinal inflammation [2,3,5,6,[9][10][11]14,17,[45][46][47]. Interestingly, human regulatory T cells, compared to CD4 + or CD8 + T cells, were shown to be exquisitely sensitive to the effects of specific PI3Kδ inhibition in terms of proliferation and suppressive function [57]. Another newly described immune deficiency/inflammatory condition termed activated PI3Kδ syndrome (APDS) has emerged and contributed additional insight into the immunoregulatory role of PI3Kδ in human diseases [58]. Patients with APDS have a gain of function mutation in the gene encoding PI3Kδ (PIK3CD) and present with recurrent bacterial and herpesvirus sinopulmonary infections, lymphadenopathy, and autoimmune/inflammatory manifestations. It is tempting to speculate that patients with APDS demonstrate opposing immune phenotypes to that of patients with IBD, due to suppression of normal bactericidal/antiviral immune responses at mucosal surfaces. Ultimately, whether through inadequate pathogen clearance (in APDS) or prolonged inappropriate inflammatory response (in IBD), these two conditions both lead to aberrant chronic inflammation and its sequelae. These data suggest that defective PI3Kδ signaling in response to resident bacteria might influence regulatory immune cells to a greater degree than effector immune cells in the intestine and other mucosal surfaces regularly in contact with resident microbes.
We also emphasize the importance of colonic lamina propria (cLP) IL-10-producing cells in intestinal homeostasis. cLP cells demonstrated markedly higher production of IL-10 and regulatory activity (IL-10/IL-12p40 ratio) compared with MLN and spleen cells in response to bacterial stimulation. The colon is an unique environment where constant exposure to resident bacteria and their products requires dedicated regulation via IL-10 to maintain homeostasis. Likewise, the colon may be more sensitive to defective IL-10 signaling compared with other organs. Our data showed that dysregulated immunity in PI3Kδ D910A mice predominantly develops in the colon through diminished IL-10 production. PI3Kδ D910A mice develop colitis characterized by microbiota-dependent Th1/Th17 cell expansion [12,31,32] despite low IFN-γ production by antigen-stimulated PI3Kδ D910A T cells [59]. This discrepancy remains unclear, but our co-culture assays revealed that defective PI3Kδ signaling in B cells promoted low IL-10 and high IL-12p40 production, impaired suppression of IL-12p40 secretion by bacteria-stimulated macrophages, and promoted naïve T cell proliferation. Since IL-12p40 promotes differentiation of naïve T cells into Th1/Th17 cells [24,60], prolonged APC stimulation conditions (high IL-12p40 and low IL-10) by PI3Kδ D910A B cells may promote expansion of Th1/Th17 cells. Moreover, IL-12p40 and the Th1 cytokine IFN-γ normally induce anti-inflammatory cytokines from T cells and B cells and regulatory cells as a negative feedback [40,45,[61][62][63], but IL-10-producing regulatory B cells are not appropriately induced to make IL-10 in PI3Kδ D910A mice (age older than 14 weeks). These findings indicate that homeostatic IL-10-production by B cells and IL-10 + regulatory B cell numbers are not adequately induced under excessive inflammatory conditions in the absence of PI3Kδ signaling, which likely worsens mucosal inflammation. Together, a deregulated immune phenotype (low IL-10 and high IL-12p40) of PI3Kδ D910A B cells appears to contribute to the pathogenesis of T cell-mediated colitis.
One of the limitations of the present study is that we did not assess IL-10-independent regulatory factors of PI3Kδ B cells, which include TGF-β, IL-27, IL-35, IgA and induction of regulatory T cells [6,[9][10][11][45][46][47]64]. However, our in vitro assays with an anti-IL-10 receptor antibody demonstrate the importance of IL-10 produced by B cells in regulating inflammatory cytokines from macrophages, T cells, and B cells themselves. Given that deficiency of IL-10, PI3Kδ, or luminal IgA affects gut microbiota colonization [65][66][67], profiling enteric microbiota in PI3Kδ D910A mice may unveil a mutual relationship between PI3Kδ function in B cells and microbial dysbiosis in IBD. An additional limitation is that we were unable to determine which bacterial species, groups or components are responsible for stimulating IL-10 through PI3Kδ in the murine B cells with our cecal lysates. This can be addressed in future studies by exploring the relative efficacy of lysates of relevant individual resident intestinal bacterial species or their components. Finally, future studies should test the ability of human fecal samples to activate PI3Kδ-dependent protective functions in human-derived colonic immune cells.
In conclusion, our study provides evidence that genetic dysfunction or pharmacological blockade of PI3Kδ in B cells lead to globally dysfunctional homeostatic pathways in the intestine and consequent development of intestinal inflammation. As PI3K signaling affects several key immune pathways, investigating cell-specific, site-specific and disease-specific PI3K-related signaling is important for better understanding of the pathogenesis of IBD and regulation of mucosal homeostasis. This knowledge may contribute to the development of novel and safe pharmacological therapies in IBD.

Supplementary Materials:
The following are available online at http://www.mdpi.com/2073-4409/8/10/1121/s1, Figure S1: B cell population is significantly reduced in PI3Kδ D910A mice, Figure S2: The inflammatory or regulatory phenotypes of MLN and spleen cells are determined by PI3Kδ signaling, Figure S3: The median fluorescence intensities of GFP in T cells and B cells, Figure S4: Phenotypic characteristics of B cells in PI3Kδ D910A mice, Figure S5: Blockade of IL-10 signaling increases IL-12p40 secretion by B cells, Table S1: Antibodies used in the present study.