Abstract
Intestinal intraepithelial lymphocytes (IEL) comprise a diverse population of cells residing in the epithelium at the interface between the intestinal lumen and the sterile environment of the lamina propria. Because of this anatomical location, IEL are considered critical components of intestinal immune responses. Indeed, IEL are involved in many different immunological processes ranging from pathogen control to tissue stability. However, maintenance of IEL homeostasis is incompletely understood. In this report we present evidence that osteopontin, a glycophosphoprotein with diverse roles in biomineralization, cell-mediated immunity, and inflammation, is important for maintaining normal levels of IEL. Mice in which the osteopontin gene (Spp-1) is disrupted present decreased levels of IEL subtypes, such as TCRαβ and TCRγδ IEL in the intestine, an effect not observed for lymphocytes in other immune compartments such as spleen or lamina propria, indicating an epithelium-specific effect. In vitro experiments show that mouse and human IEL survival is improved by culture with recombinant osteopontin. CD44, a ligand for osteopontin, is conspicuously expressed in IEL, including mucosal regulatory T cells. We present in vitro and in vivo evidence supporting a role for the osteopontin-CD44 interaction in IEL and regulatory T cell homeostasis, with implications in the development of intestinal inflammation.
Introduction
One of the largest immunological compartments in the body is comprised of intraepithelial lymphocytes (IEL), a group of immune cells interspaced between the monolayer of intestinal epithelial cells (IEC). IEL can be divided into two groups based on T cell receptor (TCR) expression.1, 2, 3 TCR+ IEL express αβ or γδ chains. TCRαβ+ IEL can be further subdivided into different populations such as TCRαβ+CD4+, TCRαβ+CD4+CD8αα+, TCRαβ+CD8αβ+, and TCRαβ+CD8αβ+CD8αα+ cells. TCRneg IEL comprise innate lymphoid cells (ILC) 4, 5, 6 and lymphocytes characterized by expression of intracellular CD3γ chains (iCD3+), some of which express CD8αα (iCD8α cells).7, 8
Because of their anatomical location, IEL function as sentinels between the antigenic contents of the intestinal lumen and the sterile environment under the basal membrane of the epithelium. Indeed, TCRγδ IEL surveil for pathogens,9 secrete antimicrobials conferring protection against pathobionts,10 and protect from intestinal inflammation.11 TCRγδ IEL are also involved in protecting the integrity of damaged epithelium after dextran sodium sulphate (DSS)-induced colitis,12 and are responsible, along with other IEL, for preserving IEC homeostasis.13, 14 Other IEL, such as conventional effector CD8 T cells that migrate into the epithelium, can protect against Toxoplasma infection,15 and reside in this organ as memory cells.16, 17 IEL such as TCRαβ+CD4+CD8αα+ cells can prevent development of colitis in an adoptive transfer model.18 TCRneg IEL such as iCD8α cells confer protection against Citrobacter rodentium infection and may protect against necrotizing enterocolitis in neonates,8 but these cells can also promote intestinal inflammation in some experimental conditions.19 iCD3+ IEL are involved in malignancies associated with celiac disease.7
Osteopontin is a glycosylated phosphoprotein, encoded by the Spp-1 gene, originally characterized as part of the rat bone matrix.20, 21 Osteopontin is a versatile molecule involved in many physiological processes, that include immunological roles, such as macrophage chemotaxis,22 induction of Th1 responses,23 suppression of T cell activated-induced cell death,24 inhibition of natural killer (NK) cell apoptosis and promotion of NK cell responses,25 and modulation of dendritic cell function.26 The role of osteopontin during intestinal inflammation is diverse. For example, Spp-1-deficient mice present with milder disease in the trinitrobenzene sulphonic acid 27 and DSS models of colitis.28 In humans with inflammatory bowel diseases (IBD), plasma osteopontin is significantly increased as compared to normal individuals.29, 30 Some reports indicate that osteopontin is downregulated in the mucosa of Crohn’s disease (CD) patients,31 whereas other groups have reported higher osteopontin expression in the intestines of individuals with CD and ulcerative colitis (UC) compared with healthy controls.29, 32 Because of its potential involvement in IBD, this molecule could be a potential biomarker for IBD,33 and has been explored as a potential therapeutic target in clinical trials.34 These reports clearly underscore the importance of osteopontin in intestinal inflammation and warrant further investigation of this molecule in mucosal immune responses.
Recently, it was reported that the frequency and number of TCRγδ IEL were reduced in osteopontin-deficient mice, while TCRαβ IEL numbers remained similar in comparison to wild type controls.35 However, in vitro neutralization of IEL-derived osteopontin resulted in decreased survival of TCRγδ and TCRαβ IEL,35 confounding the in vivo results. Moreover, because of the IEL diversity present in the intestine, more detailed analysis is required to determine the survival requirements of different IEL populations. Herein, we provide substantial in vitro and in vivo evidence indicating that osteopontin promotes the survival of TCRγδ and TCRαβ IEL, including subpopulations of these cells, such as: TCRαβ+CD4+, TCRαβ+CD4+CD8αα+ and TCRαβ+CD8αα+ cells. We also show that the survival effect is not only confined to murine IEL, but osteopontin also promotes the survival of human IEL. Additionally, osteopontin promotes a pro-apoptotic transcriptional profile, underscoring its role as a survival stimulus. Moreover, we show that the effect of osteopontin in IEL survival is mediated by CD44, a known receptor for osteopontin conspicuously expressed by IEL and regulatory T cells in the intestinal mucosa. Lastly, using two different models of intestinal inflammation, we present evidence indicating that the lack of osteopontin results in increased disease susceptibility.
Results
Osteopontin-deficient mice contain a reduced IEL compartment
To determine the role of osteopontin in IEL homeostasis, we analyzed the IEL compartment of wild-type (WT) and Spp-1−/− mice. Flow cytometry studies revealed a reduction in the proportion of total IEL in the small intestine of Spp-1−/− mice compared with WT mice (Fig. 1a, FSC vs SCC dot plots). The percentages of certain TCRβ+ IEL subpopulations, such as TCRβ+CD4+ and TCRβ+CD4+CD8αα+ were reduced in Spp-1−/− mice, whereas others, such as TCRβ+CD8αα+ cells were similar among both groups of mice (Fig. 1a dot plots). The percentages of TCRγδ+ and TCRneg IEL, such as iCD8α cells, were similar between WT and Spp-1−/− mice. In terms of total cell numbers, Spp-1−/− mice presented a significant decrease in TCRγδ+ IEL, corroborating the observations made by others (Fig 1a graphs).35 However, we observed total cell number reduction in TCRβ+ IEL due to the decline of TCRβ+CD4+, TCRβ+CD8α+ and TCRβ+CD4+CD8α+ in the small intestine IEL of osteopontin-deficient mice. The numbers of TCRneg IEL and the subpopulation iCD8α cells were similar among WT and Spp-1−/− mice (Fig 1a graphs). While colon IEL numbers from Spp-1−/− mice showed a similar pattern as in the small intestine, the numbers of TCRneg IEL from these mice were significantly reduced as compared with WT mice (Fig. 1b). Interestingly, osteopontin-deficiency did not affect spleen T lymphocytes (Fig. 1c), or lamina propria CD19+, TCRβ+CD4+ and TCRβ+CD8+ cells (Fig. 1d), suggesting that the major influence of this molecule is confined to the intestinal IEL compartment.
Increased apoptosis and decreased cellular division in IEL from osteopontin-deficient mice
To investigate whether the reduction in IEL numbers in Spp-1−/− mice was due to increased cell death, we determined the levels of early/late apoptosis and necrosis. As shown in Fig. 2a, all IEL populations isolated from WT and Spp-1−/− mice analyzed presented similar levels of cells in early apoptosis (annexin V+7AADneg). We observed a significantly higher percentage of late apoptotic (annexin V+7AAD+) TCRγδ+ and TCRβ+CD4+CD8α+, and a near significant higher percentage of TCRβ+CD4+ IEL from Spp-1−/− mice. Because the cell death and proliferation rates are inversely related, we investigated the proliferation frequencies in different IEL populations. TCRβCD4+, TCRβCD4+CD8α+ and TCRβ+CD8αα+ IEL derived from Spp-1−/− mice presented lower levels of dividing cells as indicated by the expression of Ki67 (Fig. 2b), corroborating the increase in apoptosis observed in IEL from osteopontin-deficient mice. However, despite the numbers of TCRγδ IEL were reduced in Spp-1−/− mice and presented an increased frequency of cells in late apoptosis, TCRγδ IEL from Spp-1−/− mice showed similar proliferation levels as cells derived from WT mice.
Overall, these results show that in the absence of osteopontin, IEL present higher apoptosis and reduced cell proliferation, which may account for the decreased IEL numbers observed in osteopontin-deficient mice.
CD44 deficiency affects the IEL compartment
CD44 is one of the receptors for osteopontin36 and is also expressed by IEL (Fig. 3a). Therefore, we reasoned that if osteopontin provides a survival signal via CD44, the absence of this receptor should also result in decreased numbers of IEL. Most IEL populations from CD44−/− mice presented a trend of decreased numbers in both the small intestine and colon (Fig. 3b and 3c) in comparison to IEL from wild type mice. The difference was significant for total colon TCRβ+ and subpopulations such as TCRβ+CD4+, and TCRβ+CD8αα+ IEL. Interestingly, TCRneg cells also presented a reduction in numbers in the small intestine and colons of CD44−/− mice. These results show similarities between the IEL compartment observed in Spp-1−/− and CD44−/− mice, suggesting a role for the osteopontin-CD44 interaction for IEL survival.
As shown in Fig.1, the effect of osteopontin in steady-state levels seems to affect only the IEL compartment. To determine whether the frequencies of other CD44+ cells in other immune compartments are affected by oseteopontin, we analyzed TCRγδ+, TCRαβ+CD4+ and TCRαβ+CD8+ T cells from the spleen. The fraction of CD44+ cells in these three populations was undistinguishable between WT and Spp-1−/− mice (Fig 3c), indicating that, at steady state levels, osteopontin does not affect the frequency of activated CD44+ spleen T cells.
Osteopontin promotes survival of IEL in a CD44-dependent manner
To further investigate the role of osteopontin in IEL survival, we used an in vitro system. For this purpose, first, we isolated total IEL (CD45+ cells from IEL preparations) from WT mice and cultured them under different conditions. CD45+ IEL cultured in media alone resulted in ~30% cell survival after 24 h of culture, followed by a constant decrease thereafter. However, addition of recombinant osteopontin resulted in improved IEL survival, to around 50% 24 h post culture (Fig. 4a). Recombinant osteopontin maintained increased IEL survival compared with media alone at 48 and 72 h post culture. However, when IEL were cultured in the presence of recombinant osteopontin and anti-osteopontin antibodies, cell survival was blunted to levels similar as observed for media alone, indicating that IEL in vitro survival was mediated by recombinant osteopontin (Fig. 4a). IEL incubated only with anti-osteopontin antibodies behaved similarly as cells cultured in media alone, suggesting that under these experimental conditions either IEL did not produce osteopontin or IEL-derived osteopontin was not a factor in cell survival. These results are in contrast to the work of Ito et al., that showed that IEL-derived osteopontin was important for their in vitro survival;35 differences in culture systems between the two groups may account for this discrepancy. To determine whether the observed osteopontin-mediated IEL survival was facilitated by CD44, IEL were cultured in the presence of recombinant osteopontin and anti-CD44 antibodies. As shown in Fig. 4a, CD44 blockage resulted in decreased IEL survival similar to that observed for media alone, especially at 48 and 72 h post-culture. Moreover, CD45+ IEL derived from CD44-deficient mice cultured with recombinant osteopontin presented similar survival as IEL cultured in media alone (Fig. 4b), underscoring the importance of the interaction between osteopontin and CD44 in IEL survival. We then investigated the survival of different FACS-enriched IEL subpopulations from WT mice when cultured in the presence or absence of recombinant osteopontin. Increased survival was observed in TCRγδ+, TCRβ+CD4+, TCRβ+CD8α+ and TCRβ+CD4+CD8α+ IEL when recombinant osteopontin was included in the media; however, the effect on survival was observed at different time points depending on the IEL population analyzed (Fig. 4c). Addition of anti-CD44 to the cultures blunted the osteopontin effect (Fig. 4c).
To determine whether osteopontin influences the survival of spleen T cells, we first incubated total splenocytes from WT mice in the presence or absence of recombinant osteopontin. As shown in Fig. 4d, survival of total spleen T cells was not affected by osteopontin. Moreover, FACS-enriched TCRβ+CD44hi spleen cells from WT and Spp-1−/− mice cultured in the presence or absence of osteopontin and/or anti-CD44 presented no difference in their survival (Fig. 4e), indicating that osteopontin preferentially influences the in vitro survival of IEL via CD44 but not of total or CD44+ spleen T cells.
The immune system of mice maintained in specific pathogen-free conditions more closely resembles that of human neonates rather than adults.37 Therefore, to determine whether our findings with murine IEL are relevant to humans, we isolated total IEL from human neonates and cultured them in the presence or absence of recombinant osteopontin. Human IEL survived better in the presence of recombinant osteopontin than in its absence, and addition of anti-CD44 blunted the cytokine effect (Fig. 4f), in parallel to the results observed with mouse IEL. To determine the effect of osteopontin on other human lymphocytes, we employed PBMC from healthy adults, as we were unable to obtain PBMCs from the same neonate individuals for this purpose. As shown in Figure 4g, PBMC survival was not enhanced or reduced by any of the treatments used, which corroborates an intestinal IEL specific effect. Our results indicate that osteopontin promotes survival of murine and human IEL, and at least in the mouse system, this effect is mediated by CD44.
Osteopontin induces a survival program in IEL
The in vivo and in vitro studies presented in the previous sections indicate that osteopontin is an important cytokine involved in the survival of IEL. To investigate whether osteopontin, or its absence, alters the IEL transcription profile, we isolated RNA from FACS-enriched TCRγδ+, TCRβ+CD4+, TCRβ+CD8α+ and TCRβ+CD4+CD8α+ IEL derived from naïve WT and Spp-1−/− mice, and determined the expression of genes involved in preventing apoptosis. Comparison of genes expressed in IEL from WT and Spp-1−/− mice showed that TCRγδ+ cells from WT animals had more differentially expressed anti-apoptotic genes in comparison to the other IEL populations (Fig. 5a). TCRβ+CD8α+ IEL presented little differential expression among the anti-apoptotic genes analyzed. On the other hand, TCRβ+CD4+ and TCRβ+CD4+CD8α+ IEL differentially expressed some of these genes (Fig. 5a). Birc2, a known inhibitor of apoptosis in malignancies,38 was one of the genes consistently differentially expressed in most IEL analyzed from WT mice, including TCRβ+CD4+CD8α+ cells (Fig. 5b). Other anti-apoptotic genes with higher expression in WT IEL were Prdx2, Polb, Dffa, Bric5 and Bcl10. Overall, these results indicate that osteopontin induces the expression of anti-apoptotic genes, but the gene profile varies between different IEL populations.
Because addition of recombinant osteopontin rescued wild type-derived CD45+ IEL survival when cultured in vitro (Fig. 4a), we interrogated whether addition of this cytokine in cultured wild type IEL modifies their gene expression profile. For this purpose, we cultured FACS-enriched CD45+ IEL from wild type mice in the presence or absence of osteopontin. Twenty-four hours post-culture, cells were collected, RNA extracted, sequenced and the gene expression profile determined. As recovery of sufficient cells for gene expression profile analysis after 24 h of culture from individual IEL populations was limiting, total CD45+ IEL were used as an alternative approach. Gene set enrichment analysis (GSEA) revealed that IEL cultured in the presence of recombinant osteopontin express genes associated with retinoid X receptor (RXR) functions (Fig. 5c and 5d). This set included genes encoding products such as the vitamin D receptor (VDR), which dimerizes with RXR and modulates osteopontin gene transcription by binding to its promoter region,39 and may induce a feedback loop in osteopontin-expressing IEL. GSEA also showed that IEL cultured in media alone present enriched pathways related to apoptosis, degradation of p27/p21 and downregulation of genes in Tregs (Fig. 5c and 5d). These sets included genes coding for proteasomal subunits, genes associated with cell cycle regulation (cyclin a2 and e1), and genes involved in programmed cell death pathways such as, caspases, Uba52, and Maged1. These results indicate that in vitro IEL exposure to osteopontin has an impact on the IEL gene transcription profile. However, it is important to consider that use of total CD45+ IEL as the source for RNA increases variability in the results due to the different IEL populations present in the CD45+ compartment.
Osteopontin serves as checkpoint for development of intestinal inflammation
We reasoned that if osteopontin deficiency affects the IEL compartment, and proper homeostasis of these cells is critical for protection against inflammation, then Spp-1−/− mice may be susceptible to spontaneous intestinal inflammation. For these purposes we monitored female Spp-1−/− mice from the time of weaning until 32 weeks of age. These mice gained weight during the observation period but not as much as wild type control females (Fig. 6a) and presented normal small intestine (Fig. 6b) and colon (not shown) architecture without signs of inflammation. However, spontaneous inflammation may become evident if another molecule involved in IEL homeostasis is disrupted. For this purpose, we crossed Spp-1−/− mice with thymus leukemia (TL) antigen deficient mice. TL is expressed in IEC and preferentially binds to CD8αα homodimers on IEL, controlling their effector functions and proliferation.40 While TL-deficient animals do not develop spontaneous intestinal inflammation, when crossed to a susceptible strain, the offspring present an early onset and increased incidence of spontaneous intestinal inflammation.41 Therefore, it is possible that disruption of two systems involved in IEL homeostasis, the TL-CD8αα and the osteopontin-CD44, may result in spontaneous intestinal inflammation. Spp-1−/−TL−/− mice also gained weight during all the observation period but at lower levels than WT and Spp-1−/− mice (Fig. 6a; for figure clarity, statistical significance at the relevant time points is presented in the figure legend). Interestingly, analysis of ileum pathology showed an increase in IEL and lamina propria inflammatory foci in Spp-1−/−TL−/− in comparison to WT and Spp-1−/− mice (Fig. 6b and 6c). These results indicate that in the proper context, the absence of osteopontin may lead to spontaneous intestinal inflammation, underscoring its importance as an intestinal checkpoint.
Osteopontin prevents intestinal inflammation but not migration into the epithelium
We have provided evidence indicating that the IEL deficiency observed in Spp-1−/− mice is due to IEL survival; however, it is possible that osteopontin also affects migration of T cells into the epithelium. To test this hypothesis, we adoptively transferred total spleen T cells from WT mice into Rag-2−/− or Spp-1−/−Rag-2−/− recipient mice, and after 7 days we determined the number of cells migrating into the intestinal epithelium. Both TCRβ+CD4+ and TCRβ+CD8α+ cells migrated similarly into the epithelium of Rag-2−/− or Spp-1−/−Rag-2−/− recipient mice (Fig. 7a), indicating that osteopontin does not influence the migration of these cells into the intestinal mucosa. We did not analyze spleen-derived TCRγδ cells because their numbers in the inoculum were very low and these cells do not reconstitute the mucosa properly. Interestingly, reconstitution analysis at 28 days post transfer showed a reduction in the total number of TCRβ+CD4+ and TCRβ+CD4+CD8α+ cells (Fig. 7b), which resembled what was observed in Spp-1-deficient mice (Fig. 1a). On the other hand, the numbers of adoptive transferred TCRβ+CD8α+ cells recovered were similar between Rag-2−/− or Spp-1−/−Rag-2−/− recipient mice at 28 days post transfer (Fig. 7b). To prevent the development of intestinal inflammation in Rag-2−/− recipient mice, we transferred total T cells, which includes regulatory T cells. To our surprise, Spp-1−/−Rag-2−/− recipient mice lost more weight than Rag-2−/− mice (Fig. 7c) and presented increased colon inflammation (Fig. 7d), suggesting that the absence of osteopontin in the host promoted disease development. To test whether CD44, as a receptor for osteopontin, was also involved in disease development in this system, we adoptively transferred total T cells from CD44−/− donor mice into Rag-2−/− and Spp-1−/−Rag-2−/− recipient mice. Rag-2−/− mice that received total spleen T cells from WT mice did not lose weight, whereas Rag-2−/− and Spp-1−/−Rag-2−/− recipient mice that received spleen cells from CD44−/− donor mice lost weight comparably starting at 2 weeks post transfer (Fig. 7e), with clear signs of intestinal inflammation (Fig. 7f).
Overall, this evidence indicates that adoptive transfer of total T cells into Spp-1−/− Rag-2−/− mice result in similar TCRβ+CD4+ and TCRβ+CD8α+ T cell migration into the IEL compartment at 7 days after transference; however, the total number of TCRβ+CD4+ and TCRβ+CD4+CD8α+ T were significantly reduced in an osteopontin-deficient environment. Unexpectedly, decreased IEL reconstitution was accompanied by intestinal inflammation. These results suggest that host-derived oseopontin and expression of CD44 on the transferred T cells, are important for preventing development of intestinal inflammation even in the presence of regulatory T cells.
Osteopontin sustains Foxp3 expression in Tregs
We expected that transferring total T cells into Spp-1−/−Rag-2−/− mice would result in protection against intestinal inflammation similar to that observed for Rag-2−/− recipient mice, due to the presence of regulatory T cells in the inoculum. However, because disease was observed in Spp-1−/−Rag-2−/− recipient mice, we investigated the fate of regulatory T cells in the presence or absence of osteopontin. Regulatory T cells are known to express CD44, which when ligated, promotes sustained Foxp3 expression.42 Thus, we hypothesized that binding of osteopontin to CD44 is a potential signal that maintains proper Foxp3 expression. To test this possibility, we cultured CD4 T cells derived from the intestinal mucosa from RFP-Foxp3 mice in the presence or absence of recombinant osteopontin, with or without anti-CD44. After 72 h of culture, there was an increase in the percentage of RFP+ cells in the presence of osteopontin, which was blunted with the addition of anti-CD44 antibodies (Fig. 8a). Figure 8b shows the combined fold increase over the untreated cells.
To test whether osteopontin sustains Foxp3 expression in vivo, we sorted splenic RFP+ cells from RFP-Foxp3 reporter mice and adoptively transferred them into Rag-2−/− or Spp-1−/−Rag-2−/− recipient mice. Eight weeks after transfer, IEL were isolated and the percentage of donor-derived (CD45+TCRβ+CD4+) RFP+ cells was determined (Fig. 8c, dot plots). Rag-2−/− mice presented a trend of higher percentage of donor-derived cells in the IEL compartment than Spp-1−/−Rag-2−/− recipient mice (Fig 8d). Approximately 10% of the donor-derived cells from Rag-2−/− recipient mice remained RFP+, whereas only 4% of cells recovered from Spp-1−/−Rag-2−/− recipient mice remained RFP+ (Fig. 8e). These results indicate that osteopontin sustains Foxp3 expression in regulatory T cells in the IEL compartment, possible mediated by CD44 ligation, with significant impact in the development of intestinal inflammation.
Discussion
Intestinal IEL reside in the unique environment of the IEC monolayer. In this anatomical location, IEL are poised as the first immunological layer of defense against potential pathogens from the intestinal lumen. In order for IEL to fulfill their immunological roles, they need to remain in their niche and survive. However, because IEL represent a diverse population of lymphoid cells, requirements for their homeostasis within the epithelium may depend on the particular type of IEL. For example, TCRγδ+ IEL require IL-7 for their proper development whereas other IEL are not affected by this cytokine.43 On the other hand, IL-15 deficiency does not disturb TCRγδ+ IEL but has a significant impact on TCRαβ+CD8αα+, iCD8α and iCD3+ IEL.7, 8, 44 The results presented in this report indicate that osteopontin provides survival signals to a great fraction of IEL, including TCRαβ, TCRγδ and TCRneg cells. This implies that despite having different developmental pathways and cytokine requirements, the presence of osteopontin in the epithelium ensures the survival of most types of intestinal IEL.
Osteopontin-mediated T cell survival has been documented previously. For example, concanavalin A-activated T cells from lymph nodes show reduced levels of cell death in the presence of osteopontin.24 Moreover, Hur et al. also demonstrated that osteopontin alters the expression of pro-apoptotic molecules such as Bim, Bak and Bax, promoting T cell survival.24 Using an in vitro system, we showed that TCR+ IEL rapidly die in the absence of osteopontin, whereas IEL cultured in the presence of osteopontin presented increased in vitro survival (Fig. 4). It is important to notice the differential behavior of cultured IEL subpopulations, e.g., the survival of TCRγδ+ and TCRβ+CD4+CD8α+ IEL is less than 50% after 24 h post culture decreasing to around 10% by 72 h, and it is at this point that the effect of osteopontin is more evident for these cells. On the other hand, TCRβ+CD4+ and TCRβ+CD8α+ IEL have a better survival after 24 h (more than 50%), whereas the osteopontin effect is evident for the former cells starting at 48 h. These results indicate that each IEL subpopulation possess different survival kinetics, but appear to have a similar requirement for osteopontin for their survival.
Because IEL are considered to be in a “semi-activated” state,45 osteopontin appears to primarily affect activated T cells, which express CD44. Interestingly, splenic CD44+ T cell numbers are similar in wild type and Spp-1−/− mice (Fig. 3c), suggesting that osteopontin does not affect the overall effector T cell population. Moreover, in vitro survival of CD44+ spleen T cells is not affected by addition of osteopontin (Fig. 4e). It is important to note that previous reports have demonstrated a pivotal role for osteopontin as an enhancer for the survival of effector Th17 cells, particularly during brain inflammation;24 however, whereas this group studied differentiated Th17 cells in the context of brain inflammation our results are based on CD44+ T cells in naïve animals.
In the adoptive transfer experiments reported here, donor CD4 T cells from wild type mice reconstituted the IEL compartment of Spp-1−/−Rag-2−/− recipient mice less efficiently than in Rag-2−/− recipient mice (Fig. 7), suggesting that an environment capable of producing osteopontin is important for proper cell survival. However, transfer of T cells from osteopontin-deficient donor mice into Rag-2−/− recipient mice resulted in reduced survival rates in the spleen and lymph nodes in comparison to donor T cells from wild type donor mice.25 These results indicate that intrinsic T cell-derived osteopontin is critical for normal cell reconstitution in secondary lymphoid organs whereas T cells present in the IEL compartment present an increase dependency for their survival on osteopontin derived from the environment.
Adoptive transfer of total spleen T cells into immunodeficient hosts, such as Rag-2−/− mice, results in cellular reconstitution and protection from T cell-mediated colitis due to the presence of regulatory T cells.46, 47 Surprisingly, when recipient Rag-2−/− mice were deficient in osteopontin (Rag-2−/−Spp-1−/− animals), mice developed colitis even in the presence of regulatory T cells, indicating that environmental osteopontin is important for maintaining regulatory T cell function (Fig. 7). This was evident when transfer of regulatory T cells (RFP-Foxp3+ cells) into Spp-1−/−Rag-2−/− hosts resulted in lower recovery of RFP+ cells from the intestinal epithelium in comparison to cells transferred into Rag-2−/− hosts (Fig. 8c and d). Therefore, similar to other IEL, regulatory T cells appear to be responsive to osteopontin via CD44, but in this case osteopontin helps to sustain the levels of Foxp3 expression. Of note, activation of naïve T cells (CD4+CD45RBhi) or their subsequent pathogenic capacity when adoptively transferred into Rag-2−/− mice requires T cell-derived osteopontin.48
If a significant fraction of IEL require osteopontin for their survival, what are the cellular sources for this cytokine in the intestines? Under steady-state conditions IEC do not express osteopontin, but some IEL populations do. Osteopontin expression appears to be confined to TCRγδ and TCRαβ+CD8α+ IEL.35 On a per cell basis, iCD8α cells present high levels of osteopontin mRNA expression.8 Therefore, IEL survival may depend on IEL-derived osteopontin, suggesting possible interactions between different IEL populations.
In the past few years, the role of osteopontin in the etiology of human diseases has been greatly appreciated. For example, recent work has investigated the use of neutralizing anti-osteopontin antibodies as a therapeutic with preclinical studies currently underway (ref. in 49). Studies such as the one described herein show that osteopontin neutralization may carry unwanted side-effects, especially if patients are immunocompromised. Osteopontin appears to be a critical molecule with multiple effects, one of them supporting proper IEL homeostasis, and therefore additional studies are needed to better understand its function and how it affects the biology of the mucosal immune system.
Materials and Methods
Mice
CD44−/−, Rag-2−/−, Spp-1−/− and RFP-Foxp3 reporter mice on the C57BL/6 background were obtained from the Jackson Laboratories. TL−/− mice were developed in our laboratory as previously described.41 To homogenize the microbiome of vendor-derived mice, we co-housed or bred mice with established WT C57BL/6 mice from our colony. Spp-1−/−Rag-2−/− mice were generated in our colony by breeding Spp-1+/− with Rag-2+/− mice. Spp-1−/−TL−/− mice were generated in our colony by breeding Spp-1+/− with TL+/− mice. Mice were maintained in accordance with the Institutional Animal Care and Use Committee at Vanderbilt University.
IEL isolation
IEL were isolated by mechanical disruption as previously reported.41 Briefly, after flushing the intestinal contents with cold HBSS and removing excess mucus, the intestines were cut into small pieces (~1cm long) and shaken for 45 minutes at 37°C in HBSS supplemented with 5% fetal bovine serum and 2 mM EDTA. Supernatants were recovered and cells isolated using a discontinuous 40/70% Percoll (General Electric) gradient. To obtain lamina propria lymphocytes, intestinal tissue was recovered and digested with collagenase (187.5 U/ml, Sigma) and DNase I (0.6 U /ml, Sigma). Cells were isolated using a discontinuous 40/70% Percoll gradient.
Human samples
The Vanderbilt University Medical Center Institutional Review Board approved sample collection (IRB# 090161 and 190182). All samples were de-identified and written informed consent was obtained. Peripheral blood mononuclear cells were isolated by ficoll gradient from unidentified healthy adult volunteers as previously described.50 A pathologist from the Vanderbilt Children’s Hospital provided fresh intestinal tissue specimens from infants. Isolation of human cells associated with the intestinal epithelium was performed as previously described.51 Briefly, tissue was cut in small ~1 cm pieces and incubated with slow shaking for 30 minutes at room temperature in HBSS (without calcium and magnesium) supplemented with 5% fetal bovine serum, 5mM EDTA and an 1% antibiotic mix (pen-strep-AmphoB; Fisher-Lonza). After incubation, cells in the supernatant were recovered.
Reagents and flow cytometry
Fluorochrome-coupled anti-mouse CD4, -CD44, -CD45, -CD8α, -TCRβ, -TCRγδ, Ki69 and isotype controls were purchased from Thermofisher or BD Biosciences. Annexin V and 7AAD were purchased from BD biosciences. All staining samples were acquired using a FACS Canto II or 4-Laser Fortessa Flow System (BD Biosciences) and data analyzed using FlowJo software (Tree Star). Cell staining was performed following conventional techniques. Manufacturer’s instructions were followed for Annexin V staining. FACS sorting was performed using a FACSAria III at the Flow Cytometry Shared Resource at VUMC.
In vitro survival assay
Total IEL enriched for CD45+ cells using magnetic beads (Miltenyi) or FACS-enriched subpopulations were incubated in a 96-well flat-bottom well plate (Falcon, Fisher Scientific) at a density of 5×105 cells/ml in RPMI containing 10% fetal bovine serum. In some groups culture media was supplemented with anti-osteopontin (2 μg/ml) (R&D), recombinant osteopontin (2 μg/ml) (R&D), or anti-CD44 (5 μg/ml)(Thermofisher). Cells were cultured in 5% CO2 at 37°C. At time 0 and every 24 h, an aliquot from the culture was taken to count live cells. Percentage of live cells was calculated in reference to time 0. For human samples, total PBMC or IEL were cultured in the presence or absence of recombinant human osteopontin (2 μg/ml) (R&D) and anti-human-CD44 (5 μg/ml) (Thermofisher).
Transcription profile analysis
For gene expression array, RNA was isolated from FACS-enriched IEL subpopulations from 4 individual WT and Spp-1−/− mice. Samples were prepared for RT2 Profiler PCR Array (QIAGEN PAMM-012Z) and analyzed following manufacturer’s instructions. For RNAseq analysis, RNA was isolated from FACS-enriched CD45+ IEL derived from WT mice cultured for 24 h in the presence or absence of recombinant osteopontin using the QIAGEN RNeasy micro kit. Sequencing was performed on an Illumina NovaSeq 6000 (2 x 150 base pair, paired-end reads). The tool Salmon 52 was used for quantifying the expression of RNA transcripts. The R project software along with the edgeR method 53 was used for differential expression analysis. For gene set enrichment analysis (GSEA), RNAseq data was ranked according to the t-test statistic. The gene sets curated (C2), GO (C5), immunological signature collection (C7) and hallmarks of cancer (H) of the Molecular Signatures Database (MSigDB) were used for enrichment analysis. GSEA enrichment plots were generated using the GSEA software 54 from the Broad Institute with 1000 permutations.
Adoptive transfer of total T cells
Total splenocytes from WT mice were depleted of CD19-positive cells using magnetic beads (Miltenyi). Four to 6 million cells were adoptively transferred (i.p.) into Rag-2−/− or Spp-1−/−Rag-2−/− mice. Starting weight was determined prior to injection. Seven or 28 days later, recipient mice were weighed, sacrificed and donor cells from the intestines analyzed by flow cytometry. In some experiments a segment of the colon was excised and prepared for histological examination. In some experiments CD19-depleted splenocytes from CD44−/− mice were adoptively transferred into Rag-2−/− or Spp-1−/−Rag-2−/− mice. Mice were weighed weekly for 4 weeks, and cells and colon analyzed as indicated above.
In vitro and in vivo Foxp3 expression
Lamina propria lymphocytes isolated from RFP-Foxp3 mice were cultured in the presence or absence of recombinant osteopontin and anti-CD44 antibodies as described above. At time 0 and 72 h later, cells were analyzed by flow cytometry to detect RFP expression in live TCR+CD4+ cells. For in vivo experiments, CD4+RFP+ splenocytes were enriched by FACS and 2 x105 cells were adoptively transferred i.p. into Rag-2−/− or Spp-1−/−Rag-2−/− mice. Eight weeks later, IEL were isolated and RFP expression analyzed in CD45+TCRβ+CD4+ donor-derived cells.
Statistical analysis
Statistical significance between 2 groups was determined using Mann-Whitney U-test. For analysis of 3 groups or more, one-way nonparametric (Kruskal-Wallis) test or two-way ANOVA followed by Dunn’s multiple comparison tests were used appropriately. All data was analyzed in GraphPad Prism 7 and showed as mean ± standard error mean (SEM). A P value <0.05 was considered significant.
Author Contributions
A.N., designed and performed experiments, analyzed data and wrote the manuscript; M.J.G., designed and performed experiments, analyzed data and wrote the manuscript; K.L.H., performed experiments; M.B.P., provided expert pathological analysis of colon and small intestine tissue; J-H.W., provided procurement of human samples; D.O-V., designed and performed experiments, analyzed data, wrote the manuscript and procured funding.
Acknowledgments
We thank the Flow Cytometry Shared Resource for technical help and guidance; the Translational Pathology Shared Resource for tissue processing. We thank Dr. Luc Van Kaer for revising the manuscript. This work was supported by NIH grant R01DK111671 (to D.O-V.); Careers in Immunology Fellowship Program from the American Association of Immunologist (to D.O-V. and A.N.); National Library of Medicine T15 LM00745 grant (to M.J.G.); and scholarships from the Digestive Disease Research Center at Vanderbilt University Medical Center supported by NIH grant P30DK058404.