The ontogeny of Butyrophilin-like (Btnl) 1 and Btnl6 in murine small intestine

Murine Butyrophilin-like (Btnl) 1 and Btnl6 are primarily restricted to intestinal epithelium where they regulate the function of intraepithelial T lymphocytes. We recently demonstrated that Btnl1 and Btnl6 can form an intra-family heterocomplex and that the Btnl1-Btnl6 complex selectively expands Vγ7Vδ4 TCR IELs. To define the regulation of Btnl expression in the small intestine during ontogeny we examined the presence of Btnl1 and Btnl6 in the small bowel of newborn to 4-week-old mice. Although RNA expression of Btnl1 and Btnl6 was detected in the small intestine at day 0, Btnl1 and Btnl6 protein expression was substantially delayed and was not detectable in the intestinal epithelium until the mice reached 2–3 weeks of age. The markedly elevated Btnl protein level at week 3 coincided with a significant increase of γδ TCR IELs, particularly those bearing the Vγ7Vδ4 receptor. This was not dependent on gut microbial colonization as mice housed in germ-free conditions had normal Btnl protein levels. Taken together, our data show that the expression of Btnl1 and Btnl6 is delayed in the murine neonatal gut and that the appearance of the Btnl1 and Btnl6 proteins in the intestinal mucosa associates with the expansion of Vγ7Vδ4 TCR IELs.

Detection of anti-Btnl1 and anti-TCR Vγ 7 was achieved with APC-conjugated AffiniPure F(ab') 2 fragment donkey anti-rabbit IgG (H+ L) (Jackson ImmunoResearch, West Grove, PA) and streptavidin-APC-Cy TM 7 (BD Biosciences, San Diego, CA), respectively. Cells were gated on 7AAD or LIVE/DEAD ® Fixable Red negative cells to exclude non-viable cells, and positive staining for Btnl1 was determined by comparison with pre-immune serum. Cell samples were acquired on LSR II flow cytometer using the DIVA software (BD Biosciences, San Diego, CA), and analysis of data was perfomed using the FlowJo Software version 7.6.5 (Ashland, OR). Statistical analysis. Data were generated using GraphPad Prism version 6.04 (San Diego, CA). The unpaired two-tailed t test was used for comparison between two independent groups, while One-Way ANOVA followed by Holm-Sidak´s multiple comparisons test was applied to evaluate differences between three or more groups. Pearson correlation test was performed to determine the correlation between parameters. Differences were considered as statistically significant when p < 0.05 (* P ≤ 0.05, * * P ≤ 0.01, * * * P ≤ 0.001 and * * * * P ≤ 0.0001).

Results
Appearance of Btnl1 and Btnl6 in the small intestinal epithelium is delayed during ontogeny. At birth and at weaning, mammals are exposed to multiple novel antigens from diet and microbial colonization. To gain insight into how these events regulate the expression of Btnl1 and Btnl6, small intestinal epithelial cells isolated from newborn mice and from mice pre-and post-weaning were analyzed for Btnl1 and Btnl6 expression. Although Btnl1 and Btnl6 transcripts were detected in the small intestine of newborn animals (Fig. 1A), the mRNA expression was not reflected at the protein level by flow cytometry (Fig. 1B), or immunohistochemistry (Fig. 1C), for Btnl1, or by western blot for Btnl6 (Fig. 1D). Directly ex vivo, viable 7AAD − CD45 − small intestinal cells with side and forward scatter typical of epithelial cells showed increasing levels of surface Btnl1 during intestinal ontogeny; no protein was detected in newborn and 1-week-old pups, the protein appeared when the mice had reached the age of 2 weeks (2.4% ± 0.3) and then increased to reach levels comparable to those detected in adult animals (19% ± 5) by 4 weeks of age (24% ± 7) (Fig. 1B). Whereas flow cytometry analysis detected Btnl1 cell surface expression, the immunostaining of intestinal tissue visualized total protein levels; in 4 μ m sections of small intestinal tissue Btnl1 was first detected at 2 weeks of age and, consistent with flow cytometry data, protein expression was found to increase with increasing age (Fig. 1C). As for Btnl1, the expression of Btnl6 proteins was substantially delayed and was not detectable in the intestinal epithelium until 3 weeks of age (Fig. 1D). Thus, the appearance of Btnl proteins occurred in pre-weaning pups kept mainly on a milk diet and was not associated with weaning.
The delay in Btnl1 and Btnl6 protein appearance in the neonate gut is not associated with gut colonization. In order to determine if the expression of Btnl1 and Btnl6 in the intestinal mucosa in the early postnatal period is dependent on the presence of intestinal microbiota, crude intestinal samples from adult mice maintained under strict GF conditions were analyzed with real-time PCR for the presence of Btnl1 and Btnl6 transcripts. As is evident in Fig. 2A, Btnl1 and Btnl6 RNA was readily expressed in GF animals and the level of RNA expression, although slightly lower, was comparable to RNA levels found in animals housed in CV conditions ( Fig. 2A). To determine protein levels, small intestinal tissue derived from GF mice was immunostained with anti-Btnl1 antibody for Btnl1 detection, or used for western blot analysis for detection of Btnl6. Btnl1 + epithelial cells were detected in the intestine of GF animals with a frequency comparable to CV mice (Fig. 2B). Likewise, Btnl6 was present in lysates of small intestinal tissue of GF mice (Fig. 2C). Hence, these data show that Btnl1 and Btnl6 are expressed in the absence of intestinal microbiota and indicate that the delay in Btnl protein expression in the intestine of CV mice is not due to gut colonization.
Expansion of Vγ7Vδ4 TCR IELs in the neonate gut associates with the appearance of the Btnl1 and Btnl6 proteins. Our recent data show that Btnl1 promotes IEL proliferation and that Btnl1-Btnl6 heteromers increase the frequency of Vγ 7Vδ 4 TCR IELs in particular 6 . In view of the current results showing a delay in Btnl1 and Btnl6 protein expression in neonatal intestinal mucosa the relative frequencies of γ δ and α β TCR IELs and the relative frequencies of Vγ 7 and Vγ 1 chains, the principal chains utilized in C57BL/6 mice 24 , were determined. This analysis identified γ δ TCR T cells as the predominant intraepithelial T cell subset comprising 80% (78% ± 9) of the total IELs at birth (Fig. 3A). The ratio between γ δ :α β IELs then dramatically inverted and at day 7, and until 2 weeks of age, α β TCR T cells represented the dominant IEL subset (63% ± 8 α β TCR IELs vs. 32% ± 7 γ δ TCR IELs) (Fig. 3A). At the age of 3 weeks the frequency of γ δ TCR T cells increased substantially (58% ± 5 γ δ TCR IELs vs. 36% ± 4 α β TCR IELs) and at this age the IEL composition was similar to the composition of IEL population in adult mice (Fig. 3A). The significant enrichment of γ δ TCR T cells at week 3 coincided with the substantial increase in Btnl1 levels and the appearance of Btnl6 in the intestinal mucosa. To determine whether the appearance of the Btnl proteins coincided with increased Vγ 7Vδ 4 TCR levels, we examined the relative frequencies of Vγ 7, Vδ 4 and Vγ 1 IELs. Our data show that within the γ δ TCR IEL subset, cells expressing Vγ 7 and cells expressing Vγ 7Vδ 4 TCR increased markedly between postnatal weeks 2 and 3 (from 50% ± 5 to 78% ± 5 and from 9% ± 1 to 16% ± 4, respectively) (Fig. 3B,C), and that the expansion of these cells correlated with a substantial increase in Btnl1 protein levels (Fig. 3B,C). In contrast, the ratio of Vγ 1-bearing IELs was not specifically affected by the appearance of the Btnl proteins (Fig. 3D).

Discussion
We have analyzed the ontogeny of Btnl1 and Btnl6 in the small intestine of neonatal mice. Our data identify a substantial delay in Btnl1 and Btnl6 protein expression in the neonatal intestinal epithelium and demonstrate that these proteins are absent in the small intestinal mucosa at birth and during the first 2-3 weeks of life. The delay in Btnl1 and Btnl6 protein expression was not reflected at the RNA level suggesting post-transcriptional mechanisms that regulate Btnl protein expression in the postnatal gut. Such mechanisms may involve microR-NAs that function in RNA silencing and have the capacity to repress the translation of transcribed mRNAs 25 , or post-translational turnover of the protein. During early neonatal life, important changes occur in the intestine. The developing gut immune system is challenged by milk and microbial flora and later the diet of mice changes from milk to pelleted food leading to changes in microbial contents. This period is essential for a complete development of the mucosal immune system and we therefore assessed the impact of gut microbiota on Btnl expression by investigating the presence of Btnl1 and Btnl6 proteins in GF animals. We found that Btnl expression does not correlate with microbial exposure as mice housed in GF conditions had Btnl1 and Btnl6 protein levels which were not different from conventional mice. Although the presence of Btnl1 in 14-day-old pre-weaning pups kept mainly on a milk diet suggests that expression of Btnl proteins is not dependent on change of diet, we cannot determine whether Btnl expression is regulated by increasing exposure to dietary antigen before weaning, or if the expression is regulated by an unknown developmental factor. We previously reported that Btnl1 can promote the expansion of small intestinal IELs, and that Btnl1 in a heteromeric protein complex with Btnl6, augments the expansion of γ δ T cells bearing the Vγ 7Vδ 4 TCR in particular 6 . To examine if the appearance of the Btnl1 and Btnl6 proteins in the intestinal epithelium was associated with the expansion of γ δ TCR cells we examined the percentage of Vγ 7 and Vγ 7Vδ 4 bearing IELs in the neonatal mice. In agreement with previous reports investigating neonatal mouse small intestine 26 , we found that between 2 and 3 weeks of age γ δ TCR IELs increased substantially, whereas the percentage of α β TCR IELs decreased. Within the expanding γ δ TCR IEL compartment Vγ 7 and Vγ 7Vδ 4 TCR bearing T cells showed the greatest increase that correlated with the appearance of the Btnl proteins in the intestine. The expansion of γ δ Τ CR IELs is indicative of a major influx of γ δ TCR lymphocytes and/or a local expansion of resident γ δ TCR IELs in the neonatal gut epithelium. Observations from rat IEL ontogeny in which relative IEL numbers were similar in normal and athymic 4-8 mice/group were included and unpaired two-tailed t test was used for statistical analysis (* P ≤ 0.05, * * P ≤ 0.01, * * * P ≤ 0.001 and * * * * P ≤ 0.0001). (B) Small intestinal sections were immunostained with anti-Btnl1 rabbit polyclonal antiserum (red) and counterstained with DAPI (blue) to visualize nuclei. No staining was detected using pre-immune serum. Original magnification 20x. Four mice were stained for each group and representative stainings are shown. (C) Lysates from small intestinal tissue of GF and CV adult C57BL/6 mice (20 μ g) were analyzed for Btnl6 protein expression. Lysates from MODE-K cells transfected with FLAG-tagged Btnl6 cDNA pMX-IRES-GFP served as a positive control. The predicted protein migrating under reducing conditions at the theoretical molecular weight of ~59 kDa for FLAG-tagged Btnl6 and ~58 kDa for non-tagged Btnl6 was detected with anti-FLAG antibody or Btnl6-specific polyclonal antibody. No bands were detected on gels immunoblotted with pre-immune serum. The β -actin immunoblot acts as a loading control. Data are representative of two experiments. GF: germ-free; CV: conventional.  /group (B-D) were analyzed and One-Way ANOVA followed by Holm-Sidak´s multiple comparisons test was used for statistical analysis. Correlation between Btnl1 expression and the percentage of Vγ 7 TCR IELs (B) or Vγ 7Vδ 4 TCR IELs (C) during the mouse ontogeny was determined using the Pearson correlation test (* P ≤ 0.05, * * P ≤ 0.01, * * * P ≤ 0.001 and * * * * P ≤ 0.0001). Each dot in the correlation analysis in Fig. 3B,C represents % of Btnl1 + epithelial cells vs % of Vγ 7 (Vδ 4) TCR IELs of one mouse at a particular time-point. Newborn −8-week-old mice were used in this correlation analysis. neonates 27 , and from mouse studies in which γ δ TCR IELs were regenerated in the small intestine in the absence of the thymus 28 , suggest that the IEL colonization process may be regulated by gut micro-environmental factors rather than by immigration of thymus-derived T cells. Intriguingly, representation of Vδ 4-expressing IEL subsets is influenced by genes linked to the MHC Class II region 24,29 , which also contains the Btnl1 and Btnl6 genes. Although further experiments, for example using Btnl-/-approaches, will be necessary to confirm the association between Btnl1 and Btnl6 and the expansion of γ δ TCR IELs, particularly those bearing the Vγ 7Vδ 4 TCR, these data support our recent in vitro results 6 and further add strength to the evidence of a link between Btnl genes and the γ δ expressing IEL repertoire in the intestinal intraepithelial compartment.