T cell-specific deficiency in BBSome component BBS1 interferes with selective immune responses

Abstract

Bsardet Biedl syndrome (BBS) is a genetic condition associated with various clinical features including cutaneous disorders and certain autoimmune and inflammatory diseases pointing to a potential role of BBS proteins in the regulation of immune function. BBS1 protein, which is a key component of the BBSome, a protein complex involved in the regulation of cilia function and other cellular processes, has been implicated in the immune synapse assembly by promoting the centrosome polarization to the antigen-presenting cells. Here, we assessed the effect of disrupting the BBSome, through Bbs1 gene deletion, in T cells. Interestingly, mice lacking the Bbs1 gene specifically in T cells (T-BBS1^−/−) displayed normal body weight, adiposity, and glucose handling, but have smaller spleens. However, T-BBS1^−/− mice had no change in the proportion and absolute number of B cells and T cells in the spleen and lymph nodes. There was also no alteration in the CD4/CD8 lineage commitment or survival in the thymus of T-BBS1^−/− mice. On the other hand, T-BBS1^−/− mice treated with Imiquimod dermally exhibited a significantly higher percentage of CD3-positive splenocytes that was due to CD4 but not CD8 T cell predominance. Notably, we found that T-BBS1^−/− mice had significantly decreased wound closure, an effect that was more pronounced in males indicating that the BBSome plays an important role in T cell-mediated skin repair. Together, these findings implicate the BBSome in the regulation of selective functions of T cells.

INTRODUCTION

Bardet Biedl syndrome (BBS) is an autosomal recessive and genetically heterogeneous disorder that belongs to the larger group of disorders known as ciliopathy. BBS caused by mutations in at least 24 genes (BBS1–BBS24) (1, 2) is diagnosed based on the presence of primarily several phenotypes that include obesity, visual impairment, polydactyly, hypogonadism, renal dysfunction, and learning disabilities (3–5). Recent evidence indicates that patients with BBS are prone to cutaneous disorders and certain autoimmune and inflammatory diseases such as type 1 diabetes, thyroiditis, rheumatoid arthritis, psoriasis, and inflammatory bowel diseases, with BBS1 and BBS10 being the most frequent causative genes (6, 7). Consistent with these findings, BBS genes were found to be expressed in the lymphoid tissues (spleen and lymph nodes) as well as in T and B cells (6, 8). Moreover, Bbs4 gene-deficient mice displayed altered B cell homeostasis as indicated by the increased frequency of B cell precursors in the bone marrow and low numbers of marginal zone B cells (6).

The products of eight BBS genes (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9, and BBS18) are known to form a protein complex called the BBSome (9, 10). The BBSome plays an important role in the function of the primary cilium through its involvement in the intraflagellar transport system that mediates trafficking of cargo to and from the ciliary membrane. In addition, the BBSome has been implicated in the regulation of several other cellular processes not related to cilia (10). For instance, the BBSome has emerged as a key player in the trafficking to the plasma membrane of receptors underlying energy homeostasis, such as the leptin receptor and serotonin 5-HT2C receptor, explaining the obesity phenotype of BBS (11–13).

Emerging evidence implicates the BBSome in the assembly and function of the immune synapse, a specialized membrane domain that is functionally homologous to the primary cilium (14). During the formation of the immune synapse, BBS proteins appear to be critical for centrosome translocation toward the T cell interface with cognate antigen-presenting cells. In particular, BBS1 protein was identified as a key player in this process by connecting the 19S regulatory subunit of the proteasome to dynein for its transport to the centrosome during the early stages of immune synapse formation (8). Although current evidence demonstrates a link between BBSome and T cell immune synapse formation, the role of this system in the regulation of physiological processes is yet to be demonstrated.

Here, we explored the role of the BBSome in T cells through disruption of its essential component BBS1. For this, we tested the consequence of T cell-specific deletion of the Bbs1 gene on T cell populations and function and their recruitment and migration in vitro and in vivo. We also assessed the ramifications of BBS1 deficiency on energy and glucose homeostasis, psoriasis-like skin inflammation, wound healing, and tissue repair.

MATERIALS AND METHODS

Ethics Statement

Care of the mice used in the experiments met the standards outlined by the National Institutes of Health in their guidelines for the care and use of experimental animals. The study was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Iowa (Protocol No. 1101549). Treatments were randomized using table of random numbers and data analysis was performed blindly. Isoflurane (up to 5% for induction; 1.5%–2% for maintenance) was used as anesthetic. Mice were euthanized with CO₂ asphyxiation followed by harvest of organs (e.g., brain, liver, thymus, spleen, skin) after death.

Generation of Mice Lacking the Bbs1 Gene in T Cells (T-BBS1^−/−)

Mice expressing floxed alleles of the Bbs1 gene (Bbs1^flox/flox) reported previously (11, 15) were obtained from our colonies. ROSA (Stop^flox/flox-tdTomato) reporter transgenic mice and CD4^Cre transgenic mice were obtained from the Jackson Laboratory (Strain Nos. 007914 and 017336, respectively). To study the role of the BBSome in T cells, we generated a new transgenic mouse line in which the Bbs1 gene is selectively deleted in T cells. We first crossed Bbs1^{flox/flox,ROSA} mice that were generated previously (11, 12, 16) with CD4^Cre mice to generate Bbs1^+/flox,ROSACD4^Cre mice. The resulting mice were subsequently backcrossed with Bbs1^{flox/flox,ROSA} mice to generate the Bbs1^{flox/flox,ROSA}CD4^Cre mice (referred to as T-BBS1^−/−). Mice were genotyped by polymerase chain reaction (PCR) analysis of tail DNA as described previously (11, 12).

All mice used in this study were from mixed 129SvEv and C57B/6J backgrounds. Adult male and female mice (8–16 wk of age) were used unless indicated otherwise. Mice were housed in groups of three to five per cage and maintained on 12-h light-dark cycle with lights on at 6:00 AM. Room temperature was maintained at 22°C. Food and water were available ad libitum except when the mice were fasted as described later.

Reverse Transcription PCR

Total RNA was extracted from ∼100 mg of whole tissue (brain, liver, spleen, and thymus) using the RNeasy spin columns (RNeasy Mini Plus Kit, QIAGEN) following the manufacturer’s instructions. The RNA concentration was determined using a NanoDrop ND-1000. Using 1 μg of total RNA template, cDNA was generated using SuperScript (Invitrogen). The Bbs1 sequence was subsequently PCR-amplified with S18 used as internal control. Then, 10 µL of cDNA and 0.4 mmol/L of primers (see Supplemental Table S1, all Supplemental Material is available at https://doi.org/10.6084/m9.figshare.21578856) were added in a final volume of 50 µL Platinum Hot Start PCR 2X Master Mix (Invitrogen), and amplified in a T100 Thermal Cycler PCR System (Bio-Rad). The PCR conditions for all genes were as follows: denaturation for 5 min at 95°C, then 32 cycles for 30 s at 95°C and 30 s at 62°C and 40 s at 72°C, then 5 min at 72°C.

In a separate experiment, thymic tissue was harvested from 8- to 12-wk-old male and female transgenic and littermate controls. Thymocytes were isolated as described later and were single-cell-sorted based on tdTomato expression using fluorescence-activated cell sorting (FACS) (Becton Dickinson FACS Aria II). Total RNA isolation, cDNA generation, and PCR amplification using Bbs1 and S18 primers were carried out as described earlier.

Body Weight and Composition

In normal chow-fed male and female mice, body weight was measured at 16 wk of age. Obesity was obtained by feeding a cohort of 4-wk-old male mice a high-fat high sucrose diet (Research Diets Inc., D09071702) until 12 wk of age with body weight measured once weekly. Body composition (fat and lean masses) was determined at the end of the study by nuclear magnetic resonance imaging using a Bruker Minispec.

Glucose Tolerance Test and Insulin Tolerance Test

For the glucose tolerance test, 14- to 16-wk-old male mice were fasted overnight, and blood obtained from the tail was used to measure glucose at baseline (time 0), and then at 15, 30, 60, and 120 min after intraperitoneal injection of d-glucose (2 g/kg; Sigma-Aldrich, Cat. No. G8270). To obtain blood, a 1–2 mm piece of tissue was cut from the tail tip distal to the bone with sharp scissors. The tail was then gently massaged to produce blood (1–2 μL), which was collected directly on a glucose test strip (ONETOUCH Ultra). For the insulin tolerance test, mice were fasted for 5 h. After baseline glucose levels were measured, mice were treated with intraperitoneal insulin (0.5 U/kg; Novolin, Novo Nordisk) and blood glucose was measured again at 15, 30, 60, and 120 min after injection.

Imiquimod Treatment

The backs of 8- to 11-wk-old male mice were shaved. A total of 83.3 mg of commercially available Imiquimod 5% cream (Perrigo Pharmaceuticals) was applied to the shaved back for six consecutive days. This translates to a daily dose of 4.17 mg of the active compound. Control mice were treated similarly with a control cream (Vaseline Deep Moisture Cream, Unilever).

Histological Analysis

Skin samples from the back (∼1.0 × 1.0 cm) were immersed in O.C.T compound medium (Invitrogen), flash-frozen in liquid nitrogen and 2-methylbutane, and stored at −80°C until use. Seven-micrometer cryosections of snap-frozen skin were cut using a cryostat (Leica Microtome RM2135). The slides were then sequentially stained using filtered hematoxylin and eosin (H&E), followed by dehydration in ascending alcohol solutions, cleared with xylene, and cover-slipped with Permount. The slides were then examined on an Olympus IX-71 microscope at ×5 and ×20 magnification and then converted to. TIFF digital image files using CellSense Olypus Software Suite. The .TIFF files were then exported into ImageJ processing suite, available as a free download from the National Institutes of Health. The polygon section tool was then used to outline the area of interest and measure the length of the dermal/epidermal junction, the surface of the epidermis, as well as the total area of the epidermis as previously described (17).

Wound Healing Assay

For in vivo wound healing assay, isoflurane-anesthetized 9- to 11-wk-old male and female T-BBS1^−/− mice and littermate controls were subject to a 2-mm, full-thickness punch biopsy on their shaved backs using a stainless-steel biopsy punch instrument (WPI, Model 500077) that creates two wounds (left and right). The rate of right wound closure by secondary intention healing was assessed. For this, digital imaging was used to assess wound area just after the biopsy (day 0) and then on day 4 and day 7. Wound edges were found with ImageJ-Fiji algorithm (FindEdges) and wound areas were measured using ImageJ-Fiji scale and measurement tools. Wound closure on day 4 and day 7 is expressed as percentage of wound area remaining relative to the original dimensions at day 0 (wound healing rate) (18).

Flow Cytometry

The thymus, spleen, and lymph nodes (bilateral inguinal lymph nodes were used) were harvested from 8- to 12-wk-old male and female mice and minced through 70-μm mesh to obtain single-cell suspensions in radioimmunoprecipitation (RIPA) buffer modified with 2% fetal bovine serum. The spleens were treated with ammonium-chloride-potassium (ACK) lysis buffer to deplete erythrocytes. The cells were washed twice before 2–3 × 10⁶ cells, per staining, were incubated at 4°C for 30 min with various antibodies conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin-chlorophyll-protein (PerCP), allophycocyanin (APC), or cyaninine-5 (Cy5) for multiple color-fluorescent staining. The following surface markers were used for cellularity analysis: CD3, CD4, CD8, and CD45R. For intracellular detection of cytokines, 2 × 10⁶ cells were stimulated with plate-bound anti-CD3 mouse antibody [10 μL/phosphate-buffered saline (PBS)] and anti-CD28 mouse antibody (10 μL/PBS) overnight at 37°C and then incubated in the presence of GolgiStop (BD Biosciences) for 4 h. The cells were then harvested and stained with anti-CD4 and anti-CD8 mouse antibodies followed by intracellular staining using mouse Alexa Fluor-700-conjugated anti-CD44 antibody and allophycocyanin-conjugated anti-interferon γ (IFN-γ) after fixation and permeabilization (BD Cytofix/Cytoperm Plus, BD Biosciences). The antibodies and fluorophores used are described in Supplemental Table S1.

Listmode data were acquired on Becton Dickinson LSRII (BD Biosciences) using DiVa software. The results were analyzed using FlowJo software (Tree Start). Forward and side light scatter gating for single lymphocytes, which excludes cell aggregates, small erythrocytes, and dead cellular debris, was used to analyze flow-cytometric data (for gating strategies, see Supplemental Figs. S1–S3). Viability of cells was also checked with Ghost Dye Violet 540 (Tonbo Biosciences).

Data Analysis

The data are expressed as means ± SE. Normal distribution of data was assessed before statistical analysis was performed using t test or repeated-measures two-way analysis of variance (ANOVA). GraphPad PRISM 9.1.0 was used for statistical analysis. A P value <0.05 was considered to be statistically significant.

RESULTS

Validation of Bbs1 Gene Deletion in T Cells

We previously generated mice homozygous for the floxed Bbs1 allele (Bbs1^flox/flox, Fig. 1A) and heterozygous for the red reporter tdTomato (Bbs1^{flox/flox, ROSA}) (11, 12). To understand the role of the T cell BBSome, we generated a mouse model lacking the Bbs1 gene specifically in T cells by crossing the Bbs1^{flox/flox, ROSA} mice with CD4^Cre mice, which leads to deletion of loxP-flanked gene segments in thymocytes at the CD4⁺CD8⁺ stage (19). The Bbs1^{flox/flox,ROSA}/CD4^Cre (T-BBS1^−/−) mice were born at the expected Mendelian ratio, and they survive and breed normally. We verified the excision of the third coding exon of the Bbs1 gene in thymocytes by genomic PCR (Fig. 1B). There was no qualitative difference in PCR product notable in spleen samples from T-BBS1^−/− versus littermate control mice, as expected, due to the relatively lower percentage of T lymphocytes in the spleen compared with the thymus. We further performed PCR on FACS-sorted tdTomato-positive and -negative thymocytes, confirming the T cell-specific deletion of the Bbs1 gene (Fig. 1C). Lack of functional antibodies to BBS1 precluded us from evaluation of protein downregulation in T cells of T-BBS1^−/− mice versus littermate controls.

Figure 1.

Validation of T cell-specific Bbs1 gene deletion in T-BBS1^−/− mice. A: schematic representation of the conditional Bbs1 gene and the targeted region of exon 3 used to validate gene deletion. B: detection of the excision of exon 3 of the Bbs1 gene in thymus and spleen by genomic reverse transcription-PCR. Liver and brain tissue were used as controls. C: verification of T cell-specific Bbs1 gene deletion in thymocytes by RT-PCR. TdTomato is expressed in Cre-dependent manner in T-BBS1^−/− mice. Fluorescence-activated cell sorting (FACS)-sorted tdTomato (TdT)-positive and -negative thymocytes were used to generate cDNA via RT PCR. The 515 bp fragment in exon3 (sense primer) and exon 8 (antisense primer) were amplified, and S18 was used as internal control. Male and female mice were used.

T Cell-Specific BBS1 Deficiency Does Not Affect Body Weight and Glucose Metabolism

Because disruption of the BBSome is associated with development of obesity and dysregulation of metabolism both in humans (20) as well as in animal models (21), we set to examine the consequence of T cell-specific BBS1 deficiency on the regulation of energy homeostasis. However, there was no difference in body weight of either male or female T-BBS1^−/− mice as compared with littermate controls (Fig. 2A). Consistent with the lack of difference in body weight, fat mass (P = 0.94 in males and P = 0.33 in females), and lean mass (P = 0.97 in males and P = 0.15 in females) were comparable between BBS1^−/− mice and controls (Supplemental Fig. S4, A–D). We next tested the possibility that challenging mice with an obesogenic diet may unmask differences in body weight. In contrast to our expectation, feeding male mice a high-fat high-sucrose diet for 8 wk beginning at weaning led to similar weight gain in both genotypes (Fig. 2B). This was further confirmed by the absence of difference in fat and lean masses (Fig. 2, C and D). These findings indicate that the T cell BBS1 is not involved in the regulation of body weight and adiposity.

Figure 2.

Bardet Biedl syndrome 1 (BBS1) deficiency does not affect body energy and glucose homeostasis. A: comparison of body weight of normal chow-fed male and female T-BBS1^−/− and littermate control (LMC) mice (16 wk old). B: effect of high-fat/high-sucrose diet on body weight in male T-BBS1^−/− and control mice (n = 9 LMC and 12 T-BBS1^−/− mice). C and D: comparison of fat mass and lean mass between T-BBS1^−/− and control male mice on high-fat/high-sucrose diet (12 wk old). E–H: glucose tolerance test (GTT) in male mice fed normal chow (E and F, n = LMC and T-BBS1^−/− mice) or high-fat/high-sucrose diet (G and H, n = 9 LMC and 12 T-BBS1^−/− mice). Area under the curve of GTT displayed in F and H. I–L: insulin tolerance test (ITT) in male mice fed normal chow (I and J, n = LMC and T-BBS1^−/− mice) or high-fat/high-sucrose diet (K and L, n = 9 LMC and 12 T-BBS1^−/− mice). Area under the curve of ITT displayed in J and L (14–16 wk old, n = 11–13). Statistical significance was determined by t test or repeated-measures two-way ANOVA with P < 0.05 considered significant.

Next, we measured the consequence of T cell BBS1 deficiency on glucose handling and insulin sensitivity in lean and diet-induced obese mice. Glucose tolerance testing showed that lean and diet-induced obese T-BBS1^−/− mice have no change in glucose handling relative to their control littermates (Fig. 2, E–H). Insulin intolerance testing revealed that lean and obese T-BBS1^−/− mice tended to be insulin resistant compared with controls, but this was not statistically significant (Fig. 2, I–L). Thus, T cell BBS1 does not appear to be involved in the regulation of energy and glucose homeostasis.

BBS1 Deficiency Does Not Alter the Thymocytes and Peripheral T Cell Populations

The thymus size of T-BBS1^−/− mice was comparable with that of littermate controls (P = 0.999 by unpaired t test, n = 16/group, both males and females). Flow cytometric analyses revealed that T-BBS1^−/− mice exhibited normal frequencies and numbers of thymocyte populations, including double-negative (CD4⁻CD8⁻), double-positive (CD4⁺CD8⁺), and CD4 (CD4⁺CD8⁻), and CD8 (CD4⁻CD8⁺) single-positive cells (Fig. 3, A and B). Thus, elimination of BBS1 from double-positive thymocytes did not affect CD4/CD8 lineage commitment or survival in the thymus.

Figure 3.

Normal thymocytes and peripheral T and B cell populations in T-BBS1^−/− mice. A: representative fluorescence-activated cell sorting (FACS) profiles of thymus showing CD4⁺ and CD8⁺ expression from 8- to 10-wk-old T-BBS1^−/− mice and littermate control (LMC) mice. [Q1: CD4, Q2: CD4⁺CD8⁺ (double positive, DP), Q3: CD4⁻CD8⁻ (double negative, DN), Q4: CD8⁺]. B: percentages (top) and numbers (bottom) of CD4⁺, CD8⁺, DN, and DP in 8- to 10-wk-old mice [n = 7 (5 males and 2 females) LMC mice and n = 5 (2 males and 3 females) T-BBS1^−/− mice]. C: spleen weight in 8- to 10-wk-old T-BBS1^−/− mice, combined or segregated by sex. D: percentages (top) and numbers (bottom) of T and B cells in the spleens and lymph nodes [n = 7 (5 males and 2 females) LMC mice and n = 5 (2 males and 3 females) T-BBS1^−/− mice]. B cells and T cells were identified by FACS analysis using B220/CD45R and CD3, respectively. *P < 0.05 vs. LMC. Statistical significance was determined by an unpaired t test with P < 0.05 considered significant.

Adult T-BBS1^−/− mice exhibited smaller spleens compared with controls. This difference was significant in the female but not in male mice (Fig. 3C). In contrast, the inguinal lymph nodes appeared normal by visual inspection, but weight was not determined due to small size.

We performed cellularity analysis using immunofluorescence and flow cytometry to determine the T and B cell populations in the spleen and the lymph nodes, respectively. The T-BBS1^−/− mice have normal proportions and absolute numbers of B cells (R45/B220⁺) and T cells (CD3⁺) in the spleen and the lymph nodes compared with controls (Fig. 3D). T-BBS1^−/− mice also displayed normal proportions of CD4 (CD4⁺CD8⁻) and CD8 (CD4⁻CD8⁺) T cells in the spleen and lymph nodes (Supplemental Fig. S5, A and B). These results demonstrate that Bbs1 gene deletion from thymocytes at the double-positive stage does not substantially affect the CD4 and CD8 T cell development, homeostasis, and maturation in secondary lymphoid organs.

BBS1 Loss Affects Selective T Cell Responses

It was previously shown in vitro that BBS1 play an important role in T cell synapse assembly and polarized vesicle trafficking, which is essential for intracellular signaling and T cell activation (8). Thus, we hypothesized that BBS1 deficiency may alter T cell function in vivo. To determine the percentage of IFNγ-positive cells in the spleen at baseline, splenic cells were activated ex vivo by plate-bound anti-CD3 and -CD28 antibodies overnight at 37°C, followed by incubation in the presence of GolgiStop for 4 h. After intercellular staining for CD44 and IFN-γ, the cells were analyzed using flow cytometry. At baseline, compared with controls, the T-BBS1^−/− mice demonstrated slightly, but not significantly, lower production of IFN-γ by both CD4 and CD8 cells (Fig. 4, A and B).

Figure 4.

Bardet Biedl syndrome 1 (BBS1) deficiency does not affect intrinsic T cell response. A and B: representative fluorescence-activated cell sorting (FACS) profiles (A) and percentages (B) of IFNγ production by CD4 and CD8+ positive splenocytes, stimulated ex vivo by plate-bound anti-CD3/CD28 antibodies from 8- to 12-wk-old T-BBS1^−/− (n = 3 males and 4 females) and littermate control (LMC, n = 2 males and 5 females) mice. Boxed indicate cells that are CD4⁺IFNγ⁺ (left) or CD8⁺IFNγ⁺ (right). Statistical significance was determined by an unpaired t test with P < 0.05 considered significant.

We next asked whether BBS1 deficiency affects recruitment and migration of T cells. For this, we used an Imiquimod-induced, psoriasis-like skin inflammation model (22). First, we used wild-type mice to establish that application of Imiquimod induces psoriasis. About 3 days after the start of Imiquimod application, the treated skin of all the mice was notable for increased erythema, thickening, and some scale formation, which considerably worsened after 6 days of treatment (Supplemental Fig. S6A). Analysis of H&E-stained sections showed dermal immune infiltrate, parakeratosis of the stratum corneum, absence of granular layer, and acanthosis of the epidermis in the skin of Imiquimod-treated mice relative to mice treated with Vaseline cream (Supplemental Fig. S6B). Imiquimod-treated mice also displayed significant increase in the weight of the spleen (Supplemental Fig. S6C). Thus, application of Imiquimod to mouse skin leads to the formation of dermatitis that closely resembles human plaque-type psoriasis with respect to erythema, skin thickening, scaling, and morphological alterations.

Because Imiquimod-induced inflammation is T cell-dependent (22), we hypothesized that if Bbs1 gene deletion affects T cells function then it will lead to reduced recruitment and/or signaling of T cells in response to Imiquimod. Thus, we applied Imiquimod to the shaved backs of T-BBS1^−/− and control mice for six consecutive days. However, no difference was noted in the degree of skin inflammation in T-BBS1^−/− mice relative to controls (Fig. 5A). H&E analysis showed that the degree of epidermal acanthosis was not different between Imiquimod-treated T-BBS1^−/− and control mice, but the dermal immune infiltrate was reduced in the skin of T-BBS1^−/− mice (Fig. 5, B and C). In addition, spleen weight tended to be lower in the Imiquimod treated T-BBS1^−/− mice relative to controls (Fig. 5D).

Figure 5.

Effect of Imiquimod on skin inflammation and T cell activation in T-BBS1^−/− mice. A: phenotypical representation of mouse back skin after 6 days of treatment of 8- to 11-wk-old male T-BBS1^−/− and littermate control (LMC) mice. B: representative sections of H&E staining of the back skin. C: epidermal thickness of skin sections from T-BBS1^−/− and control mice. The epidermis was measured using ImageJ open-source software. For each section, three independent measurements were taken and averaged to determine the epidermal thickness. D: weight of the spleen relative to body weight (BW) after 6 days of treatment with Imiquimod vs. vaseline cream. E: percentages of splenocyte CD3⁺, CD4⁺, and CD8⁺ cells. F: percentage of splenocyte CD44⁺ and IFNγ⁺ CD4 and CD8 cells as identified by fluorescence-activated cell sorting (FACS) analysis in the Imiquimod-treated control and T-BBS1^−/− mice. *P < 0.05 and **P < 0.0001 vs. LMC. Statistical significance was determined by an unpaired t test with P < 0.05 considered significant.

To determine whether there was a difference in T cell activation in response to Imiquimod treatment in T-BBS1^−/− versus control mice, splenic cells were activated ex vivo by plate-bound anti-CD3 and -CD28 antibodies as described earlier. There was a significantly higher percentage of CD3-positive splenocytes in the T-BBS1^−/− mice, and this difference was due to CD4 but not CD8 T cell predominance (Fig. 5E). Interestingly, there was no difference in the absolute degree of activation (CD44⁺) or cytokine production (IFNγ) of these cells between genotypes (Fig. 5F). Of note, the number of IFNγ producing CD4 and CD8 T cells from Imiquimod-treated mice is higher relative to untreated mice (Fig. 4). Thus, although BBS1 deficiency did not affect the processes of keratinocyte hyperproliferation and disruption of epidermal differentiation, characteristic of Imiquimod-induced psoriatic dermatitis model, it increased CD3-positive splenocytes.

BBS1 Deficiency Leads to Wound Healing Defects

We have previously shown that BBS1 hypomorphic mice, bearing a systemic homozygous M390R knock-in mutation that characteristically exhibit impaired cilia function and recapitulate the BBS phenotype, have altered fibroblast function and reduced wound healing and tissue repair (23). We hypothesized that the BBSome in T cells is critical for proper wound healing, and its disruption will result in delayed tissue repair. To test this hypothesis, we evaluated wound-healing responses to skin punch biopsy in T-BBS1^−/− and control mice. The timing of fibrin clot formation was similar between genotypes. However, as shown in Fig. 6, male T-BBS1^−/− mice had decreased wound closure that was more pronounced and statistically significant at day 7. Interestingly, female T-BBS1^−/− mice showed a trend toward reduced wound healing at 4 and 7 days as well. These results demonstrate that BBS1 plays an important role in T cell-mediated skin repair and that this effect may be sex-specific.

Figure 6.

Bardet Biedl syndrome 1 (BBS1) deficiency alter wound healing. A: average percentages of wound area remaining after 4 days and 7 days, relative to day 0 when the wound was performed, in 9- to 11-wk-old male (top) and female (bottom) littermate control (LMC) and T-BBS1^−/− mice. B: representative images showing that male littermate control (LMC) mice achieved complete wound closure of the original 2.0-mm biopsy by day 7 post wound, whereas T-BBS1^−/− male mice demonstrated significantly delayed wound healing. *P < 0.05 vs. LMC. Statistical significance was determined by an unpaired t test with P < 0.05 considered significant.

DISCUSSION

The high prevalence of certain autoimmune and inflammatory diseases in patients with BBS points to a potential role for the BBSome in the regulation of immune function (6). Consistent with such an idea, BBS genes and proteins are expressed in immune tissues and cells and the BBSome component BBS1 in the formation of the immune synapse, an essential structure for intracellular signaling and T cell activation (6, 8). Here, we show that mice lacking the Bbs1 gene specifically in T cells have normal body weight and unaltered metabolic function, but they display smaller spleens. Imiquimod-induced, psoriasis-like skin inflammation was not different between T-BBS1^−/− and control mice, but T-BBS1^−/− mice exhibited a significantly higher percentage of CD3-positive splenocytes due to CD4 T cell predominance. Moreover, the wound-healing assay revealed that T-BBS1^−/− mice have decreased wound closure at day 7, indicating that the BBSome is required for skin repair. Together, these findings implicate the BBSome in the regulation of selective T cell functions.

Lack of effect of BBS1 deficiency on T cell development, homeostasis, and maturation is consistent with previous findings demonstrating that mice lacking another BBSome component, BBS4, globally or specifically in T cells have no major alterations in the T cell compartment although global Bbs4 null mice displayed a significant decrease in percentage of T cells among the splenocytes and decreased the percentage of CD44⁺ cells among splenic CD8⁺ T cells (6). The unaltered response of T cells to activation by anti-CD3 and -CD28 antibodies in T-BBS1^−/− mice is also in line with the normal T‐cell‐mediated autoimmune response observed in mice lacking the Bbs4 gene in T cells (6). However, T cell activation in Imiquimod-treated T-BBS1^−/− mice led to significantly higher percentage of CD3-positive splenocytes. Thus, the effect of Bbs1 gene deletion on T cell function appears to be context-dependent.

Cutaneous disorders including psoriasis are common in patients with BBS (7, 24). Haws et al. (7) showed that all 21 patients with BBS analyzed display dermatologic findings. In addition, we previously demonstrated that the hypomorphic Bbs1^M390R mice have significantly delayed wound closure (23). We and others have attributed the cutaneous phenotypes of BBS to dysfunction of fibroblasts and other epidermal and dermal cells based on the well-known importance of the BBSome on the function of cilia that are present in these cells. However, our current findings implicate T cells in the dermatologic manifestations of BBS, as T cell-specific Bbs1 gene deletion recapitulated the delayed wound closure. These findings demonstrate the role of the T cell BBSome in efficient wound repair. T cells, which are known to be stimulated during tissue damage, may contribute to wound repair through interaction with other cells and by resolving inflammation and producing reparative cytokines and growth factors that promote the complex and active process of tissue repair (25, 26). It should be noted, however, that CD4 or CD8 deficiency does not interfere with wound closure (27). On the other hand, γδ T cells which are the major immune cells of skin have very different effects on wound reepithelialization, inflammation, contraction, and collagen deposition based on the cytokines and growth factors they produce (28, 29). Thus, further investigation is needed to understand the aspects of T cell function impacted by BBSome and mechanisms underlying its role in wound healing.

In contrast to the reduced wound repair, T cell Bbs1 gene deletion did not alter the Imiquimod-induced psoriatic dermatitis. This could be attributed to the fact that T-BBS1^−/− mice did not develop obesity, a condition that is thought to play an important role in driving the cutaneous phenotypes of BBS (6). It should be noted, however, that T-BBS1^−/− mice exhibit delayed wound closure, indicating that the absence of obesity is unlikely to explain why these mice did not develop an exaggerated psoriasis phenotype relative to control animals. Alternatively, the lack of effect of Bbs1 gene deletion on the processes of keratinocyte hyperproliferation and disruption of epidermal differentiation characteristic of the Imiquimod-induced psoriatic dermatitis model could be explained by the overall proportionally lower level of cytokine production displayed by lymphocytes lacking the BBS1. There were proportionally more CD4⁺ T cells in the spleens of T-BBS1^−/− mice; however, those produced similar amounts of IFNγ. Therefore, even though there seems to be an impairment in cytokine production in T-BBS1^−/− mice, there is also increased recruitment of CD4⁺ cells in these mice resulting in a similar level of inflammation compared with control mice. It remains to be determined whether the increase in CD4⁺ T cells in the spleen of Imiquimod-treated T-BBS1^−/− mice reflect their recruitment, an increase in proliferation, or a lack of egress.

T cells have profound influence on the physiological processes that determines body weight, adiposity, glucose handling, and insulin sensitivity (30–33). However, our data indicate that BBS1 in T cells is not required for the control of body weight and adiposity even when challenged with an obesogenic diet. Glucose handling was also unaffected by T cell Bbs1 gene deletion. Interestingly, there was a slight, but not statistically significant, reduction in insulin sensitivity in T-BBS1^−/− mice that tended to be pronounced in mice fed a high-fat/high-sucrose diet. Longer expose to the obesogenic diet may be necessary to reveal the potential role of T cell BBS1 in the regulation of insulin sensitivity.

Perspectives and Significance

Our findings implicate the BBSome in mediating selective T cell functions. Indeed, ablation of the BBSome component BBS1 in T cell led to proportionally more CD4⁺ T cells in the spleens and higher CD3⁺ splenocytes in Imiquimod-treated, BBS1-deficient mice. We also show that BBS1 deficiency interferes with wound healing. The connection between the BBSome and immune function can explain why patients with BBS are prone to skin and immunological diseases (6, 7). However, our findings indicate that loss of the BBSome function in T cells does not seem to underlie the high prevalence of psoriasis in patients with BBS. This does not exclude a potential role of T cell dysfunction in other cutaneous and autoimmune disorders observed in patients with BBS, but this remains to be determined. It will also be interesting to examine whether the T cell BBSome contributes to other phenotypes that are commonly associated with BBS such as renal abnormalities, retinal degeneration, and cognitive impairments. In addition, future studies are needed to understand the link between the BBSome and immune cells more precisely. Measuring other parameters of T cell activation such as proliferation or expression of CD69 or CD25 in T-BBS^−/− mice could reveal potential role of the BBSome in the stimulation of T cells. This will help decipher the mechanisms underlying the clinical features of BBS.

DATA AVAILABILITY

Data will be made available upon reasonable request.

SUPPLEMENTAL DATA

Supplemental Table S1 and Figs. S1–S6: https://doi.org/10.6084/m9.figshare.21578856.

GRANTS

This work was supported by the National Institutes of Health (NIH) T32AI007260 (to M. Stump) and NIH HL084207, Veterans Affairs (VA) I01 BX004249, VA IK6 BX006040, and the University of Iowa Fraternal Order of Eagles Diabetes Research Center (to K. Rahmouni). The University of Flow Cytometry Facility is a core resource supported by the University of Iowa Carver College of Medicine and Holden Comprehensive Cancer Center.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

M.S. and K.R. conceived and designed research; M.S. and D.F.G. performed experiments; M.S., D.F.G., and K.R. analyzed data; M.S., D.F.G., and K.R. interpreted results of experiments; M.S., D.F.G., and K.R. prepared figures; M.S. drafted manuscript; M.S., D.F.G., and K.R. edited and revised manuscript; M.S., D.F.G., and K.R. approved final version of manuscript.