Interleukin-7 Enhances the Th1 Response to Promote the Development of Sjögren's Syndrome–like Autoimmune Exocrinopathy in Mice

Authors


Abstract

Objective

Although elevated interleukin-7 (IL-7) levels have been reported in patients with primary Sjögren's syndrome (SS), the role of IL-7 in this disease remains unclear. We undertook this study to characterize the previously unexplored role of IL-7 in the development and onset of primary SS using the C57BL/6.NOD-Aec1Aec2 (B6.NOD-Aec) mouse model, which recapitulates human primary SS.

Methods

For gain-of-function studies, recombinant IL-7 or control phosphate buffered saline was injected intraperitoneally (IP) into 12-week-old B6.NOD-Aec mice for 8 weeks. For loss-of-function studies, anti–IL-7 receptor α-chain (anti–IL-7Rα) antibody or its isotype control IgG was administered IP into 16-week-old B6.NOD-Aec mice. Salivary flow measurement, histologic and flow cytometric analysis of salivary glands, and serum antinuclear antibody assay were performed to assess various disease parameters.

Results

Administration of exogenous IL-7 accelerated the development of primary SS, whereas blockade of IL-7Rα signaling almost completely abolished the development of primary SS, based on salivary gland inflammation and apoptosis, autoantibody production, and secretory dysfunction. IL-7 positively regulated interferon-γ (IFNγ)–producing Th1 and CD8+ T cells in the salivary glands without affecting IL-17. Moreover, IL-7 enhanced the expression of CXCR3 ligands in a T cell– and IFNγ-dependent manner. Accordingly, IFNγ induced a human salivary gland epithelial cell line to produce CXCR3 ligands. IL-7 also increased the level of tumor necrosis factor α, another Th1-associated cytokine that can facilitate tissue destruction and inflammation.

Conclusion

IL-7 plays a pivotal pathogenic role in SS, which is underpinned by an enhanced Th1 response and IFNγ/CXCR3 ligand–mediated lymphocyte infiltration of target organs. These results suggest that targeting the IL-7 pathway may be a potential future strategy for preventing and treating SS.

Sjögren's syndrome (SS) is a chronic autoimmune disease affecting 2–4 million Americans ([1]). The major immune-mediated pathologic events of SS are lymphocytic infiltration of salivary and lacrimal glands and production of autoantibodies, which together cause destruction and dysfunction of these exocrine glands ([1, 2]). SS patients have xerostomia and keratoconjunctivitis, which are often accompanied by systemic inflammation ([1, 2]). SS can occur in the form of primary SS or secondary SS, which is associated with other connective tissue diseases ([1, 3]). The etiology of SS is still unclear, although viral infection and genetic predisposition are implicated as likely triggers ([1, 2, 4-7]). Both autoreactive T and B cells play essential roles in the autoimmune responses that cause tissue inflammation, autoantibody production, and clinical onset of SS ([2, 3, 8-10]). T cell–derived cytokines, including interferon-γ (IFNγ), interleukin-4 (IL-4), and IL-17, exert essential but distinct functions in SS pathogenesis ([11-15]). However, signals and factors that can affect the differentiation and functions of the autoreactive effector T cells in SS remain unclear.

IL-7 is a crucial cytokine that supports T cell development and homeostasis ([16-18]). Recent research has revealed additional roles of IL-7 in promoting differentiation and functions of multiple effector T cell subsets, especially IFNγ- or IL-17–producing T cells ([19-22]). Consequently, IL-7 plays a pathogenic role in multiple autoimmune diseases, including rheumatoid arthritis (RA) ([22-24]), inflammatory bowel disease (IBD) ([25-27]), and experimental autoimmune encephalomyelitis (EAE) ([19]). The levels of IL-7 and the frequency of IL-7 receptor α-chain (IL-7Rα)–positive T cells in the salivary glands of primary SS patients are significantly higher than those in non-SS patients with sicca syndrome, and they correlate with disease severity ([28, 29]). Moreover, IL-7 treatment of peripheral blood T cells from primary SS patients increases the production of Th1 and Th17 cytokines ([28, 30]). These lines of evidence suggest a possible pathogenic role of IL-7 in primary SS.

Many studies have shown that IL-7 enhances the function and expansion of IFNγ-producing Th1 cells ([20-23, 28, 30, 31]), which constitutes a chief mechanism by which IL-7 facilitates IBD ([25-27]), RA ([21, 22]), type 1 diabetes mellitus ([18, 32]), and EAE ([19]). IFNγ promotes SS development both at the preimmunologic and the immunologic phase ([11, 13]). In addition to IFNγ, IL-7 enhances secretion of tumor necrosis factor α (TNFα) from peripheral blood T cells of RA patients ([21, 22]) and primary SS patients ([28]). TNFα is implicated in numerous inflammatory and autoimmune disorders, and its levels are elevated in SS patients ([11]). Alone or in concert with IFNγ, TNFα induces death and secretory dysfunction of salivary gland cells in vitro via multiple mechanisms ([33-36]).

In the present study, we investigated the in vivo role of IL-7 in the development and onset of primary SS using C57BL/6.NOD-Aec1Aec2 (B6.NOD-Aec) mice, a well-defined model of primary SS ([37, 38]). By using both gain- and loss-of-function approaches, we demonstrated that IL-7 plays a crucial role in the development and onset of primary SS in B6.NOD-Aec mice by enhancing Th1 response and IFNγ-dependent CXCR3 ligand expression in the salivary glands. Therefore, we defined a previously unexplored role of IL-7 in the development of primary SS–like autoimmune exocrinopathy and revealed critical underlying mechanisms.

MATERIALS AND METHODS

Mice

C57BL/6 (B6) mice, recombination-activating gene 1–deficient (RAG-1−/−) mice, and IFNγ−/− mice were purchased from The Jackson Laboratory. B6.NOD-Aec mice were from the University of Florida. Mice were kept under pathogen-free conditions. All experiments were carried out under the guidelines of the Institutional Animal Care and Use Committee at The Forsyth Institute.

Histology and immunofluorescence staining

Tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to 5 μm thickness. Sections were then stained with hematoxylin and eosin and examined for leukocyte infiltration. Some sections were subjected to deparaffinization, rehydration, and antigen retrieval. They were then incubated with phycoerythrin (PE)–conjugated anti-mouse CXCL9 (MIG-2F5.5; eBioscience) or goat anti-mouse CXCL10 (C-19; Santa Cruz Biotechnology) at 4°C overnight, followed by incubation with Alexa Fluor 488–conjugated anti-goat IgG (Invitrogen). The stained samples were examined with a confocal laser scanning microscope (Leica Microsystems). Images were average projections of 3 optical sections and were processed with Leica confocal software.

Antibodies

Cells were stained and analyzed on a FACSAria III cell sorter (Becton Dickinson), with dead cells excluded by forward light scatter. The following fluorescence-conjugated antibodies were used in addition to those mentioned above: PE- or PE–Cy7–conjugated anti-CD4 (GK1.5), allophycocyanin–Cy7– or PerCP–Cy5.5–conjugated anti-CD8α (536-7), Pacific Blue (PB)–conjugated anti–T cell receptor β (anti-TCRβ) (H57-597), PE-conjugated anti–IL-7Rα (A7R34), and PB-conjugated anti–IL-17 (TC11-18H10.1) (all from BioLegend); fluorescein isothiocyanate (FITC)–conjugated anti-CD19 (ebio1D3) and FITC-conjugated anti-IFNγ (XMG1.2) (both from eBioscience); and anti-human CXCL9 (B8-11) and PE-conjugated anti-human CXCL10 (6D4/D6/G2) (both from BD PharMingen). Purified monoclonal rat anti-mouse IL-7Rα (A7R34) and its isotype control rat IgG2a (2A3) were from BioXcell.

Preparation of single-cell suspensions

Submandibular salivary glands, submandibular lymph nodes (LNs), or spleen were cut into small fragments, placed in a grinder, and processed with a tissue homogenizer. Tissue homogenates were filtered through a 100 μm nylon mesh, washed, and purged of erythrocytes with ACK lysing buffer. The single cells were resuspended in culture medium.

Ex vivo T cell stimulation and intracellular cytokine staining

Single cells prepared from various organs were stimulated in vitro with phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (1 μM) (both from Calbiochem) for 4 hours, with the addition of monensin (eBioscience) in the final 2 hours. Cells were then stained for surface markers and intracellular cytokines with intracellular cytokine staining kits (eBioscience, BioLegend) following the manufacturers' instructions.

Real-time reverse transcriptase–polymerase chain reaction (RT-PCR).

Total RNA was reverse-transcribed into complementary DNA (cDNA) using oligo(dT) and Moloney murine leukemia virus RT (Promega). The cDNA was subjected to real-time PCR amplification (Qiagen) for 40 cycles, with annealing and extension temperature at 60°C, on a LightCycler 480 Real-Time PCR System (Roche). Primer sequences were as follows: mouse IL-7, 5′-GGAACTGATAGTAATTGCCCG-3′ (forward) and 5′-TTCAACTTGCGAGCAGCACG-3′ (reverse); IFNγ, 5′-GGATGCATTCATGAGTATTGC-3′ (forward) and 5′-CTTTTCCGCTTCCTGAGG-3′ (reverse); IL-17, 5′-GCGCAAAAGTGAGCTCCAGA-3′ (forward) and 5′-ACAGAGGGATATCTATCAGGG-3′ (reverse); TNFα, 5′-CCTTTCACTCACTGGCCCAA-3′ (forward) and 5′-AGTGCCTCTTCTGCCAGTTC-3′ (reverse); mouse CXCL9, 5′-CCCTCAAAGACCTCAAACAGT-3′ (forward) and 5′-AGCCGGATCTAGGCAGGTT-3′ (reverse); and mouse CXCL10, 5′-CCAGTGAGAATGAGGGCCAT-3′ (forward) and 5′-CCGGATTCAGACATCTCTGC-3′ (reverse). Other sequences will be provided upon request from the corresponding author.

Enzyme-linked immunosorbent assay (ELISA).

Mouse IL-7 (BioLegend) concentration in serum and human CXCL9 (PeproTech), CXCL10 (R&D Systems), and IL-7 (BioLegend) concentrations in supernatants from in vitro human salivary gland (HSG) cell cultures were determined using ELISA kits according to the manufacturers' protocols.

In vivo administration of anti–IL-7Rα antibody and recombinant human IL-7.

Female B6.NOD-Aec mice were injected intraperitoneally (IP) with 100 μg of control IgG or anti–IL-7Rα 3 times weekly, starting from age 16 weeks. For in vivo IL-7 administration, female B6.NOD-Aec mice were injected IP with 5 μg recombinant human IL-7 (Biological Resources Branch, National Cancer Institute) 3 times weekly for 8 weeks, starting from age 12 weeks. Mice were then killed and their organs were harvested for analysis.

In situ apoptosis detection

Paraffin-embedded tissue sections were deparaffinized, hydrated, and then subjected to in situ apoptosis assay using a Trevigen TACS.XL In Situ Apoptosis Detection kit (purchased from R&D Systems) according to the manufacturer's instructions. Briefly, rehydrated tissue sections were partially digested with proteinase K for 20 minutes, and then endogenous peroxidases were inactivated by incubation in 3% H2O2. DNA fragmentation was then detected following the manufacturer's protocol.

Detection of serum antinuclear antibodies (ANAs).

ANAs in mouse sera were detected using HEp-2 cell substrate slides (Inova Diagnostics). Briefly, fixed HEp-2 cell substrate slides were overlaid with 1:40-diluted mouse sera and incubated for 1 hour at room temperature in a humidified chamber. After washes with phosphate buffered saline (PBS), the substrate slides were incubated with 1:100-diluted Alexa Fluor 568–conjugated goat anti-mouse IgG (Invitrogen) for 1 hour at room temperature. After PBS washes, the slides were analyzed under an FSX100 fluorescence microscope (Olympus) at 200× magnification. All images were obtained with FSX-BSW software with constant exposure of 0.1 second (Olympus).

Measurement of salivary flow rate

Nonanesthetized mice were weighed and given an IP injection of 100 μl PBS- based secretagogue solution containing isoproterenol (0.02 mg/ml) and pilocarpine (0.05 mg/ml). One minute after secretagogue injection, saliva was collected with a micropipette continuously for 5 minutes from the oral cavity of mice. The volume of saliva was measured and normalized to the body weight.

In vitro culture and treatment of HSG cells

HSG cells (0.1 × 106) were seeded in each well of a 6-well culture plate and incubated in the presence or absence of recombinant human IFNγ (50 ng/ml) or recombinant human IL-7 (50 ng/ml) for 1 or 3 days, with monensin added in the last 4 hours of culture. The supernatants were collected for ELISA, and the cells were analyzed for intracellular chemokines.

Statistical analysis

All statistical significance was determined by Student's t-test (2-tailed, 2-sample equal variance). P values less than or equal to 0.05 were considered significant.

RESULTS

Characterization of infiltrating T cells and IL-7 expression in salivary glands of B6.NOD-Aec mice

B6.NOD-Aec mice are B6 mice that carry Idd3 and Idd5 genetic segments derived from NOD mice and develop human primary SS–like exocrinopathy ([37, 38]). Previous studies showed that B6.NOD-Aec mice develop lymphocytic infiltration in exocrine glands at age 12–16 weeks and substantial secretory dysfunction at age 16–20 weeks ([13, 37]). Since the disease course and severity may vary among different animal facilities, we first examined the time course of disease development in female B6.NOD-Aec or control B6 mice belonging to 3 age groups, 10–12, 16–18, and 24 weeks.

No inflammation was observed in submandibular salivary glands from control B6 mice in any age group (further information is available upon request from the corresponding author). No inflammation was detected in the submandibular salivary glands of B6.NOD-Aec mice at age 10–12 weeks or age 16–18 weeks, but clear leukocyte infiltration and foci were detected at age 24 weeks (further information is available upon request from the corresponding author). Flow cytometric analysis showed that submandibular salivary glands from B6.NOD-Aec mice contained markedly higher percentages of TCRβ+ T cells and CD19+ B cells than did control B6 mice at age 24 weeks (Figure 1A). Within submandibular salivary gland–infiltrating mononuclear cells, significantly higher proportions of CD4+ and CD8+ T cells and CD19+ B cells were observed in B6.NOD-Aec mice compared to control mice (Figure 1B), with the majority of T cells being IL-7Rα positive (Figure 1B). Significant proportions of CD4+ and CD8+ T cells produced IFNγ, and a small proportion of CD4+ T cells produced IL-17 (Figure 1B). The proportions of T and B cells in submandibular gland draining LNs were comparable between B6.NOD-Aec and control mice, but CD4+ and CD8+ T cells from B6.NOD-Aec mice contained markedly higher percentages of IFNγ- and IL-17–positive cells (further information is available upon request from the corresponding author).

Figure 1.

Elevated interleukin-7 (IL-7) levels in C57BL/6.NOD-Aec1Aec2 (B6.NOD-Aec) mice. A, Percentage of T cell receptor β (TCRβ)–positive or CD19+ cells among total submandibular salivary gland (SMX) cells from C57BL/6 (B6) control mice or B6.NOD-Aec mice. B, Top left and Middle, Flow cytometry of lymphocyte populations in submandibular salivary gland–infiltrating mononuclear cells and intracellular cytokines in CD4+ and CD8+ T cells and CD19+ B cells from 24-week-old B6 control mice or B6.NOD-Aec mice. Top right, Percentages of CD4+ and CD8+ T cells among mononuclear cells from both groups of mice. Bottom, Percentages of interferon-γ (IFNγ)–positive or IL-17–positive T cells among mononuclear cells in submandibular salivary glands. C, Left, Real-time polymerase chain reaction analysis of IL-7 mRNA levels in submandibular salivary glands from B6 control mice or B6.NOD-Aec mice, presented relative to that of β-actin. Right, Serum levels of IL-7 measured by enzyme-linked immunosorbent assay. Data are representative or the average of analyses of at least 6 mice for each group (2 mice per experiment, 3 independent experiments). Values are the mean ± SEM. IL-7Rα = IL-7 receptor α-chain.

Since IL-7 can enhance both Th1 and Th17 responses ([20-22, 31]), and IL-7 levels are elevated in primary SS patients ([28]), we focused our attention on IL-7. Simulating human primary SS patients, B6.NOD-Aec mice exhibited markedly higher IL-7 messenger RNA (mRNA) levels in submandibular salivary glands and higher IL-7 concentrations in sera than control mice in all age groups examined (Figure 1C). Moreover, IL-7 levels in B6.NOD-Aec mice increased significantly between ages 10–12 weeks and ages 16–18 weeks (Figure 1C). Thus, B6.NOD-Aec mice developed SS-like lymphocytic infiltration in the salivary glands, which was accompanied by elevated IL-7 levels.

Administration of exogenous IL-7 enhances Th1 response and accelerates development and onset of primary SS

To assess whether administration of IL-7 could facilitate the development of primary SS, we administered 5 μg recombinant human IL-7 IP into B6.NOD-Aec mice, 3 times weekly starting from age 12 weeks. After 8 weeks of IL-7 treatment, we examined the mice for various disease parameters of primary SS. Histologic analysis showed leukocyte infiltration in submandibular salivary glands of IL-7–treated mice, whereas no such infiltration was detected in PBS- treated controls (Figure 2A). In situ assay for tissue apoptosis showed that IL-7 administration substantially aggravated apoptosis of submandibular salivary glands (Figure 2B). Analysis of serum ANAs against a human epithelial cell line, HEp-2, showed that IL-7 treatment markedly increased the percentages of mice that were serum ANA positive, as indicated by the presence of antibodies against HEp-2 substrates in the serum detected by a fluorescence-conjugated anti-mouse IgG antibody (Figure 2C). Both nuclear and cytoplasmic staining patterns were detected, consistent with a previous report ([39]). IL-7–treated mice showed a significantly decreased saliva flow rate upon pilocarpine stimulation as compared to control mice (Figure 2D), indicating an exacerbated secretory dysfunction. Hence, IL-7 administration accelerated the development and onset of primary SS.

Figure 2.

Exogenous IL-7 administration accelerates the development and onset of primary Sjögren's syndrome. Phosphate buffered saline (PBS) or recombinant human (rh) IL-7 was administered to 12-week-old B6.NOD-Aec mice 3 times weekly for 8 weeks. A, Left, Hematoxylin and eosin staining of submandibular salivary gland sections. Right, Focus score of immune cell foci in submandibular salivary glands. B, In situ TUNEL staining of submandibular salivary gland sections for detection of tissue apoptosis. C, Left, Detection of serum antinuclear antibodies (ANAs). Right, Percentages of mice that were ANA positive. D, Stimulated saliva flow rate normalized to body weight. E, Percentages of lymphocyte populations among total submandibular salivary gland cells. F, Percentages of IFNγ- or IL-17–positive T cells among mononuclear cells in submandibular salivary glands. G, Real-time polymerase chain reaction analysis of gene expression in submandibular salivary glands and draining lymph nodes (dr LN), presented relative to that of β-actin. Data are representative or the average of analyses of 9 mice for each group (3 mice per experiment, 3 independent experiments). Values are the mean ± SEM. Original magnification × 40. TNFα = tumor necrosis factor α (see Figure 1 for other definitions).

We next assessed the changes in inflammatory T cell subsets and cytokines induced by IL-7 treatment. Significantly more mononuclear leukocytes, as determined by forward and side scatter profiles, were present in the submandibular salivary glands of IL-7–treated mice than in those of PBS-treated controls (further information is available upon request from the corresponding author). The percentages of CD4+ and CD8+ T cells and CD19+ B cells among submandibular salivary gland–infiltrating mononuclear cells (further information is available upon request from the corresponding author) and their percentages among total submandibular salivary gland cells (Figure 2E) were markedly increased by IL-7 treatment. The frequencies of CD4+ and CD8+ T cells that produced IFNγ in submandibular salivary glands, spleen, and draining LNs were elevated by IL-7 treatment (further information is available upon request from the corresponding author). The percentages of IFNγ-positive T cells among total mononuclear cells in the submandibular salivary glands, spleen, and draining LNs were all significantly increased by IL-7 treatment (Figure 2F) (further information is available upon request from the corresponding author).

In comparison, IL-7 treatment did not increase in a statistically significant manner either the proportion of T cells that produced IL-17 (further information is available upon request from the corresponding author) or the frequency of IL-17–positive T cells among the total submandibular salivary gland–infiltrating mononuclear cells (Figure 2F). Compared to control mice, IL-7–treated mice had higher amounts of IFNγ, TNFα, and T-bet mRNA in submandibular salivary glands and draining LNs (Figure 2G), whereas IL-17 mRNA levels were not affected (Figure 2G). Hence, exogenous IL-7 administration preferentially increased Th1 responses in the target tissues and accelerated the development and onset of primary SS.

Blockade of IL-7Rα reduces Th1 response and inhibits development and onset of SS

We next assessed whether blockade of endogenous IL-7 activity can inhibit disease development by administration of a nondepleting monoclonal anti–IL-7Rα antibody, previously shown to inhibit IL-7R signaling in vivo ([40, 41]). We injected 100 μg of anti–IL-7Rα or its isotype control IgG IP into B6.NOD-Aec mice 3 times weekly, starting from age 16 weeks. After 8 weeks, we measured various disease parameters. Histologic analysis showed substantial leukocyte infiltration into submandibular salivary glands in IgG-treated control mice, which were age 24 weeks at the time of analysis (Figure 3A). In comparison, leukocyte infiltration was barely detectable in anti–IL-7Rα–treated mice (Figure 3A). IL-7Rα blockade significantly diminished apoptosis of submandibular salivary gland tissues (Figure 3B). We particularly examined the tissue areas with no obvious leukocyte infiltrations, and the apoptotic cells included both acinar cells and ductal cells of salivary glands (Figure 3B). IL-7Rα blockade also drastically reduced the percentages of mice that were serum ANA positive (Figure 3C). Finally, IL-7Rα blockade markedly improved the salivary flow rate (Figure 3D). Therefore, blockade of endogenous IL-7R signaling impeded the development and onset of primary SS.

Figure 3.

IL-7Rα blockade inhibits development and onset of primary Sjögren's syndrome. Control IgG or anti–IL-7Rα antibody was injected into 16-week-old B6.NOD-Aec mice 3 times weekly for 8 weeks. A, Left, Hematoxylin and eosin staining of submandibular salivary gland sections. Right, Focus score of immune cell infiltration in submandibular salivary glands. B, In situ TUNEL staining of submandibular salivary gland sections for detection of tissue apoptosis. C, Left, Detection of serum antinuclear antibodies (ANAs). Right, Percentages of mice that were ANA positive. D, Stimulated saliva flow rate normalized to body weight. E, Percentages of lymphocyte populations among total submandibular salivary gland cells. F, Percentages of IFNγ- or IL-17–positive T cells among mononuclear cells in submandibular salivary glands. G, Real-time polymerase chain reaction analysis of gene expression in submandibular salivary glands and draining lymph nodes (dr LN), presented relative to that of β-actin. Data are representative or the average of analyses of 9 mice for each group (3 mice per experiment, 3 independent experiments). Values are the mean ± SEM. Original magnification × 40. See Figure 1 for other definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.38007/abstract.

Further analysis showed that IL-7Rα blockade significantly reduced the percentages of mononuclear leukocytes in the submandibular salivary glands (further information is available upon request from the corresponding author). It also reduced the percentages of CD4+ and CD8+ T cells and CD19+ B cells in submandibular salivary gland–infiltrating mononuclear cells (further information is available upon request from the corresponding author) and their percentages among total submandibular salivary gland cells (Figure 3E). The proportions of CD4+ and CD8+ T cells that produced IFNγ, in both submandibular salivary glands and lymphoid tissues, were reduced by anti–IL-7Rα treatment (further information is available upon request from the corresponding author). The percentages of IFNγ+ T cells among submandibular salivary gland–infiltrating mononuclear cells were markedly decreased by IL-7Rα blockade (Figure 3F).

In comparison, IL-7Rα blockade did not significantly affect IL-17–positive T cells in submandibular salivary glands (Figure 3F) or in lymphoid tissues (further information is available upon request from the corresponding author). Consistent with these results, compared to control B6 mice, B6.NOD-Aec mice had higher mRNA levels of IFNγ, TNFα (not shown), and T-bet in submandibular salivary glands, and this increase was completely abolished by IL-7Rα blockade (Figure 3G) (further information is available upon request from the corresponding author). In contrast, the IL-17 mRNA level, which was considerably higher in B6.NOD-Aec mice than in control B6 mice, was not affected by IL-7Rα blockade (Figure 3G). Hence, blockade of endogenous IL-7 activity in B6.NOD-Aec mice preferentially reduced Th1 responses at target sites.

IL-7 is a critical factor for the development and homeostasis of naive T cells. We found that our anti–IL-7Rα treatment regimen did not significantly reduce the overall T cell numbers in either spleen or draining LNs (Figure 4A). Hence, this anti–IL-7Rα treatment regimen prevented the development and onset of SS and reduced Th1 responses at the target sites without affecting overall T cell numbers in lymphoid organs. Importantly, IL-7Rα blockade did not affect the numbers of FoxP3+CD4+ T cells, which are Treg cells, in either draining LNs or spleen (Figure 4B). Moreover, FoxP3 mRNA levels in draining LNs were not affected by IL-7Rα blockade (Figure 4B). Hence, the effects of IL-7 on Th1 responses and SS development are not caused by altered numbers of Treg cells. Thus, blockade of endogenous IL-7R signaling inhibits Th1 responses and prevents the development and onset of primary SS.

Figure 4.

Anti–IL-7Rα treatment does not affect the numbers of total T cells or Treg cells in draining lymph nodes (dr LN) and spleen. Control IgG or anti–IL-7Rα antibody was injected into 16-week-old B6.NOD-Aec mice 3 times weekly for 8 weeks. A, Absolute numbers of CD4+ and CD8+ T cells in spleen and draining lymph nodes (n = 5 mice). B, Absolute numbers of FoxP3+ Treg cells in spleen and draining lymph nodes (left) and FoxP3 mRNA levels in draining lymph nodes (right). Data are the average of analyses of 9 mice (3 mice per experiment, 3 independent experiments). Values are the mean ± SEM. See Figure 1 for other definitions.

IL-7 up-regulates CXCR3 ligand expression in submandibular salivary glands in an IFNγ- and T cell–dependent manner

IFNγ-producing T cells and natural killer cells express chemokine receptor CXCR3 ([42-44]). The ligands for CXCR3 include CXCL9, CXCL10, and CXCL11, which are often elevated in the target tissues in autoimmune settings to facilitate recruitment of effector T cells ([42-44]). Since IFNγ is a potent inducer of CXCR3 ligands ([42-44]), we postulated that IL-7–induced up-regulation of IFNγ in submandibular salivary glands would enhance CXCR3 ligand expression. We therefore administered recombinant human IL-7 IP into B6 mice and found that IL-7 treatment increased IFNγ, CXCL9, and CXCL10 gene expression in submandibular salivary glands 1 day later, while having no effects on CXCL11 (Figure 5A and data not shown). CXCR3 mRNA levels were also increased by IL-7, probably as a result of elevated expression of its ligands (Figure 5A). IL-7 did not up-regulate CXCL9 and CXCL10 expression in IFNγ−/− mice (Figure 5B), indicating that IL-7 requires IFNγ to up-regulate these CXCR3 ligands in submandibular salivary glands. Furthermore, IL-7 treatment did not up-regulate IFNγ and CXCR3 ligand expression in RAG-1−/− mice (Figure 5C), indicating that T cells were the major source of IFNγ in response to IL-7. These results indicated that IL-7 could rapidly up-regulate CXCR3 ligands in submandibular salivary glands in an IFNγ- and T cell–dependent manner.

Figure 5.

Administration of IL-7 promotes CXCR3 ligand expression in submandibular salivary glands. A–C, Real-time polymerase chain reaction analysis of gene expression in submandibular salivary glands of B6 mice (A), IFNγ−/− mice (B), or recombination-activating gene 1–deficient mice (C) 24 hours after injection of recombinant human (rh) IL-7 or phosphate buffered saline (PBS). D, CXCR3 ligand mRNA levels in submandibular salivary glands from the same B6.NOD-Aec mice treated with PBS or recombinant human IL-7 described in Figure 2. E, Immunofluorescence staining for CXCL9 (red) or CXCL10 (green) in submandibular salivary glands from mice described in D. F, CXCR3 ligand mRNA levels in submandibular salivary glands from B6 control mice and IgG- or anti–IL-7Rα–treated B6.NOD-Aec mice described in Figure 3. G, Immunofluorescence staining for CXCL9 (red) or CXCL10 (green) in submandibular salivary glands from mice described in F. Data are the average of analyses of 6–9 mice for each group (2–3 mice per experiment, 3 independent experiments). Values are the mean ± SEM. Original magnification × 80. See Figure 1 for other definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.38007/abstract.

We next assessed whether IL-7 administration also induces similar events in B6.NOD-Aec mice in SS. Indeed, B6.NOD-Aec mice that were treated with recombinant human IL-7 for 8 weeks had markedly higher levels of CXCL9 and CXCL10 mRNA in submandibular salivary glands than did PBS-treated controls (Figure 5D). Immunofluorescence staining also showed higher levels of CXCL9 and CXCL10 proteins in submandibular salivary glands of IL-7–treated B6.NOD-Aec mice (Figure 5E). Conversely, IL-7Rα blockade in B6.NOD-Aec mice reduced the amount of CXCL9 and CXCL10 mRNA (Figure 5F) and protein (Figure 5G) in submandibular salivary glands to levels similar to those observed in control B6 mice. Thus, IL-7 enhanced CXCR3 ligand expression in the submandibular salivary glands of both B6.NOD-Aec and B6 mice in an IFNγ- and T cell–dependent manner. We reasoned that IL-7 does this by enhancing the levels of T cell–derived IFNγ.

IFNγ potently induces CXCR3 ligand expression in HSG epithelial cells

CXCR3 ligands can be produced by a variety of immune cells and nonimmune tissue cells. To directly determine the effect of IL-7 and IFNγ on the production of CXCR3 ligands by salivary gland epithelial cells, we treated a human salivary gland (HSG) cell line, with IL-7 or IFNγ in vitro and measured expression of CXCR3 ligands. ELISA showed that IFNγ, but not IL-7, greatly increased the concentration of CXCL9 and CXCL10 in HSG cell culture supernatants after 1 day or 3 days of treatment (Figure 6A and data not shown). This is consistent with the findings in mice, in that the effect of IL-7 on CXCR3 ligands requires IFNγ. Furthermore, IFNγ treatment markedly increased the amount of CXCL9 and CXCL10 mRNA (Figure 6B) and protein (Figure 6C) in HSG cells, as determined by RT-PCR, flow cytometric analysis of intracellular protein, and immunofluorescence staining. Hence, IFNγ, but not IL-7, can induce expression and production of CXCR3 ligands by HSG epithelial cells.

Figure 6.

Interferon-γ (IFNγ) induces expression of CXCR3 ligands in human salivary gland (HSG) cells. HSG cells were incubated with or without IFNγ for 1 or 3 days. A, Enzyme-linked immunosorbent assay of CXCL9 and CXCL10 concentrations in culture supernatants. B, CXCL9 and CXCL10 mRNA levels. C, Left, Flow cytometry of intracellular CXCL9 and CXCL10 expression. Right, Immunofluorescence staining for CXCL9 (red) and CXCL10 (blue) in HSG cells treated with phosphate buffered saline (PBS) or IFNγ for 3 days. Data are representative or the average of analyses of 6 independent samples for each group (3 samples per experiment, 2 independent experiments). Values are the mean ± SEM. Original magnification × 40. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.38007/abstract.

DISCUSSION

The present study revealed a crucial pathogenic role of IL-7 in the development and onset of primary SS–like autoimmune exocrinopathy using a well-defined mouse model of primary SS. We showed that B6.NOD-Aec mice have increased levels of IL-7 in target organs and sera, which replicates the finding in human patients. We demonstrated that IL-7 plays an indispensable role in the development and clinical onset of this disease. Mechanistically, IL-7 is essential for the aberrantly enhanced Th1 responses and IFNγ production in the target sites, which leads to subsequent up-regulation of CXCR3 ligands to facilitate lymphocytic infiltration of the target tissues.

Previous studies have shown elevated IL-7 levels in the salivary glands and saliva of primary SS patients ([28, 30]). Here we defined a crucial requirement for IL-7 in the development of primary SS and identified IL-7 as an important new player in this disease. We showed that IL-7 positively regulated the number of IFNγ-producing CD4+ and CD8+ T cells in the salivary glands and that T cells were responsible for the production of IFNγ in response to IL-7. Indeed, many studies have shown the promoting effects of IL-7 on Th1 responses ([20-23, 31]). Since IFNγ has been shown to promote both early and late pathogenic events in SS ([11, 13]), the positive regulation of IFNγ is perhaps a chief mechanism by which IL-7 facilitates the development of primary SS. We further demonstrated that IL-7–induced IFNγ enhanced the expression of CXCR3 ligands in the salivary glands, thereby facilitating the recruitment of more IFNγ-producing T cells. Hence, IL-7, IFNγ, CXCR3 ligands, and the recruitment of CXCR3+ IFNγ-producing T cells form a positive feed-forward loop in the exacerbation of salivary gland inflammation and other subsequent pathologic changes.

Importantly, our anti–IL-7Rα treatment regimen did not affect the physiologic levels of IL-7R signaling that are required for normal T cell homeostasis, but selectively inhibited the excessive IL-7R signaling associated with SS conditions. Thus, this regimen provides the foundation for future development of therapeutic strategies that will ablate excessive IL-7R signaling without compromising overall immunity. Furthermore, IL-7Rα blockade did not affect the numbers of Treg cells in the lymphoid organs, and therefore, the promoting effects of IL-7 on Th1/Tc1 responses and SS development are not consequences of altered Treg cell numbers.

It has been shown that CXCL9 and CXCL10 are expressed in the salivary glands of primary SS patients but not healthy individuals ([45]), which was confirmed by our own studies (data not shown). The main cell types producing CXCR3 ligands in submandibular salivary glands in response to IL-7–induced IFNγ require further characterization. We demonstrated that IFNγ can induce the production of CXCL10 and CXCL9 from an HSG epithelial cell line, consistent with a previous study showing that IFNγ induces these chemokines in HSG explants ([45]). Thus, salivary gland epithelial cells may be crucial sources of CXCR3 ligands during SS development. Future studies will address the role of CXCR3 and its ligands in SS and determine whether the pathogenic effects of IL-7 are dependent on these molecules.

In addition to IFNγ, we showed that IL-7 increased TNFα levels, corroborating previous reports that IL-7 enhances TNFα production from T cells of primary SS patients ([28]) and RA patients ([21, 22]). In vitro studies have shown that TNFα and IFNγ, alone or cooperatively, can induce apoptosis and secretory dysfunction of salivary gland cells ([11, 33-36]). Accordingly, we showed that IL-7 markedly enhanced apoptosis of salivary gland tissues, which could result from the direct action of both TNFα and IFNγ and from indirect action via general enhancement of tissue inflammation. Our future studies will determine the specific functions of IFNγ or TNFα in submandibular salivary gland apoptosis in vivo by specifically blocking these cytokines and determining whether the effects of IL-7 on submandibular salivary gland apoptosis are dependent on these cytokines.

We found that compared to its effects on Th1 responses, the effects of IL-7 on Th17 responses are much less consequential, at least in the B6.NOD-Aec mouse model. The importance of IL-7 for pathogenic IFNγ production was previously shown in multiple autoimmune diseases ([25-27]). In the case of RA, IL-7 is required for optimal IL-17 production, in addition to IFNγ ([21, 22]). Thus, the in vivo role of IL-7 can differ in different disease settings, probably as a result of varying cellular compositions and cytokine milieu.

B6.NOD-Aec mice have elevated levels of IL-7 in target organs and serum, similar to several other autoimmune disease models ([21, 46]). The regulation of IL-7 expression by external signals is not well understood. Recent studies have shown that tissue production of IL-7 can be regulated by Toll-like receptor (TLR) signals ([47]) and IFNγ ([48]). Currently, we are investigating whether IL-7 expression can be induced in the salivary glands by TLR signaling, representing one possible trigger of SS.

The present study focused on the functions of IL-7 in the development and onset of SS. Our ongoing and future studies will determine the effects of both exogenous and endogenous IL-7 on the persistence of SS after disease onset and will assess whether IL-7Rα blockade can reverse or ameliorate the established disease, which will provide the crucial basis for the future development of anti–IL-7Rα therapeutic strategies. We will also characterize the functions of IL-7 in the pathologies of lacrimal glands, the other primary target site of primary SS. Finally, the triggers and pathogenic mechanisms of primary SS are complicated and multifactorial. The various mouse models may each recapitulate part of the pathogenic pathways or one subtype of human primary SS. Therefore, the role of IL-7 needs to be further tested in other mouse models, such as Id3−/− mice ([49]), and in human cells or tissues.

In summary, the present study demonstrates crucial pathogenic functions of IL-7 in primary SS–like autoimmune exocrinopathy in a well-defined model of primary SS. This knowledge will enable us to further investigate the role of IL-7 in other models of this disease and in human samples, in order to comprehensively understand IL-7 in relation to various aspects of SS and to develop novel therapeutic strategies.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Yu had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Yu.

Acquisition of data. Jin, Kawai.

Analysis and interpretation of data. Cha, Yu.

Acknowledgments

We thank The Forsyth Institute animal facility for maintaining animals. We also thank the Biological Resources Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute for providing recombinant human IL-7.

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