IL-17 expression is increased in lupus-prone mouse-derived T cells
To address whether Th17 cells are involved in the pathogenesis of SLE and in particular lupus nephritis, we used MRL/lpr
mice which develop readily lupus-like features. Both murine strains are characterized by the expansion of a population of T cells that do not express the CD4 and CD8 markers on their surface. Interestingly, these so-called DNT have been found to express IL-17 in the kidneys of patients with lupus nephritis (5
First, we measured the expression of IL-17 as a signature cytokine of Th17 lineage in lupus-prone MRL/lpr animals. We isolated cells from both lymph nodes and spleens of eight control (MRL/MPJ) and nine lupus-prone mice (all animals were 4 mo old).
We then briefly stimulated the lymphocytes in vitro with PMA and calcium ionophore and analyzed the IL-17 expression using intracellular staining. In , we show that lymphocytes derived from the lymph nodes of MRL/lpr mice expressed IL-17 at significantly higher levels than control lymphocytes from MRL/MPJ lymph nodes. This was true for both CD4+ T cells (percentage of MRL/lpr-derived T cells expressing IL-17A vs MRL/MPJ-derived T cells expressing IL-17A: 0.33 ± 0.05 vs 0.19 ± 0.018, p < 0.01) and DNT cells (percentage of MRL/lpr-derived T cells expressing IL-17A vs MRL/MPJ-derived T cells expressing IL- 17A: 1.25 ± 0.11 vs 0.4 ± 0.05, p < 0.001).
FIGURE 1 CD3+CD4−CD8− T cells from lupus-prone MRL/lpr mice express IL-17A at a higher level than CD3+CD4+ T cells from both lupus-prone and control mice. Lymph nodes (LN) and spleens were harvested from lupus-prone MRL/lpr and control MRL/MPJ (more ...)
Similarly, splenocytes expressing IL-17A were significantly more in MRL/lpr vs MRL/MPJ () mice. Again, both CD4+ (percentage of MRL/lpr derived T cells expressing IL-17A vs MRL/MPJ derived T cells expressing IL-17A: 0.65 ± 0.05 vs 0.18 ± 0.014, p < 0.01) and DNT cells (percentage of MRL/lpr-derived T cells expressing IL-17A vs MRL/MPJ-derived T cells expressing IL-17A: 2.2 ± 0.18 vs 0.87 ± 0.1, p < 0.005) showed similar differences between lupus-prone and control mice. Upon assessing the relative expression of IL-17A between the two cell subtypes, it was apparent that the DNT cells expressed significantly more IL-17A than CD4+ T cells in both lupus-prone and control mice ( p < 0.001 for all groups).
Contrary to IL-17A production, the DNT cells from both lymph nodes and spleens of MRL/lpr and MRL/MPJ mice did not produce significant amounts of IL-2 upon stimulation for 48 h with anti-CD3 and anti-CD28 Abs (IL-2 secretion by MRL/lpr-derived DNT cells vs MRL/lpr-derived CD4+ T cells: 10.66 ± 2.54 pg/ml vs 4373.85 ± 456.02 pg/ml, p = 0.0003). Similarly, IFN-γ was secreted at very low levels by DNT cells (IFN-γ secretion by MRL/lpr-derived DNT cells vs MRL/lpr-derived CD4+ T cells: 7.20 ± 1.33 pg/ml vs 2620.96 ± 312.7 pg/ml, p < 0.0005).
Given that lupus-prone mice differ from control mice in that the majority of T cells are DNT, we compared the two groups with regard to total number of cells expressing IL-17A. We extracted and counted the cells from both lymph nodes and spleens of nine MRL/lpr mice and eight control MPJ mice. As expected, we found that CD3+CD4−CD8− population was expanded in MRL/lpr mice (24–58% of CD3+ population, nine mice, 4-mo-old MRL/lpr mice) as opposed to control mice (1.4–6% of CD3+ population, eight mice, 4-mo-old MRL/MPJ mice). We then analyzed the expression of IL-17A in CD4+ and CD3+CD4−CD8− cells as above. In , we show that the overall number of MRL/lpr lymphocytes expressing IL-17A was significantly higher than the number of MRL/MPJ IL-17A+ lymphocytes (lymph node-derived IL-17A+ lymphocytes: MRL/lpr vs MRL/MPJ: 4,377,547 ± 90,326 vs 21,805 ± 2,883; IL-17A+ splenocytes: MRL/lpr vs MRL/MPJ: 556,401 ± 9,692 vs 19,020 ± 2,555). In addition, although both CD4+ and DNT cells expressed IL-17A in MRL/ MPJ mice (CD3+CD4−CD8− IL-17A+ vs CD4+IL-17A+ cells: 1,995 ± 406 vs 19,811 ± 2,484 in lymph nodes; 5,285 ± 855 vs 13,735 ± 2,552 in spleen; data from eight 4-mo-old MRL/MPJ animals), the MRL/lpr-derived lymphocytes had a dominant population of CD3+ CD4− CD8− IL-17A+ cells (CD3+ CD4− CD8− IL-17A+ vs CD4+ IL-17A+ cells: 3,730,215 ± 4206 vs 647,333 ± 889 in lymph nodes; 493,095 ± 9,064 vs 63,306 ± 945 in spleen; data from nine 4-mo-old MRL/lpr animals).
In addition to MRL/lpr, we evaluated the expression of IL-17A in T cells derived from both spleen and lymph nodes of another strain of lupus-prone mice, the B6/lpr mice. We found that B6/lpr lymph node-derived T cells had significantly higher expression of IL-17A when compared with control B6 mouse-derived T cells (percentage of B6/ lpr-derived CD4+ T cells expressing IL-17A vs B6-derived CD4+ T cells expressing IL-17A: 0.16 ± 0.03 vs 0.11 ± 0.01, p < 0.0002; percentage of B6/lpr-derived DNT cells expressing IL-17A vs B6-derived DNT cells expressing IL-17A: 0.38 ± 0.08 vs 0.32 ± 0.06, p < 0.035). Given the low number of DNT cells in the spleens of B6 mice, we could not measure with certainty differences between B6 DNT and B6lpr (data not shown).
In summary, IL-17A-expressing T cells are significantly increased in numbers in lupus-prone mice compared with control mice. Importantly, our results point to the fact that the major IL-17A-expressing population among T cells in the lymphoid organs of lupus-prone MRL/lpr and B6/lpr mice is the CD3+CD4−CD8− cell population.
IL-17 and IL-23 receptor expression are up-regulated in lupus-prone mouse-derived T cells
Th17 cell population maintenance and expansion relies heavily on IL-23. IL-23 binds to its receptor on the surface of T cells and leads to expression of IL-17. Because we found a higher frequency of Th17 cells in lupus-prone mice compared with control mice, we evaluated whether IL-23 is involved in the up-regulation of IL-17A in murine lupus. First, we evaluated whether the IL-23 receptor is expressed in lupus-prone mouse- derived T cells and whether its expression changes as the disease progresses. Indeed, we found that lupus-prone B6/lpr-derived lymph node cells express IL-23 receptor mRNA; this expression increased as the mouse aged and disease became more severe (, □). On the contrary, B6-derived lymph node cells express IL-23 receptor mRNA at almost undetectable levels. In addition, we found that IL-17A mRNA levels were also increased in B6/lpr mice as opposed to B6 (, □) and that the levels increased as the mice aged. Importantly, IL-23 receptor mRNA levels in these B6/lpr mice of different ages correlated with the IL-17A mRNA levels from the same animals (Pearson’s r = 0.9616, p = 0.0384, n = 4 animals/group). Contrary to this finding, IL-17F mRNA levels were not different between lupus-prone and control mice (data not shown). Second, given that the IL-23 receptor was robustly expressed at the mRNA level in lupus-prone mice, we evaluated whether treatment of cells with IL-23 would result in a further increase of IL-17A and/or expression of the IL-23 receptor. Indeed, we found that treating cells with IL-23 led to a manifold increase in the expression of IL-17A and the IL-23 receptor (). Again, IL-23 receptor mRNA showed a trend toward a statistically significant correlation with IL-17A mRNA levels (Pearson’s r = 0.9322; p = 0.0678).
FIGURE 2 IL-23 receptor and IL-17A expression is up-regulated in T cells from lupus-prone mice and correlated with disease progress. Cells were extracted from the lymph nodes of 2-mo-old B6 mice, predisease 2-mo-old lupus-prone B6/lpr, established disease 5- mo-old (more ...)
Given that the DNT population was disproportionably enriched with Th17 cells, we further evaluated whether IL-23 treatment influences CD4+ and DNT cells differently. To this end, we cultured lymph node-derived cells with anti-CD3 Ab in the presence or absence of IL-23 for 6 days and measured the expression of IL-17A. shows that T cells from either MRL/lpr or B6/lpr mice after incubation with IL-23 express higher levels of IL-17A in both DNT (percentage of IL-17A+ B6/lpr-derived T cells vs control B6: 45.2 ± 1.69 vs 18.9 ± 1.1, p < 0.0001; MRL/lpr vs control MRL/MPJ: 19.1 ± 0.52 vs 15.7 ± 0.74, p < 0.0001) and CD4+ populations than their respective controls (percentage of IL-17A+ B6/lpr-derived T cells vs control B6: 18.2 ± 1.3 vs 3.13 ± 0.04 n = 8, 5 mo old, p < 0.0001; MRL/lpr vs control MRL/MPJ: 10.7 ± 1.0 vs 6.8 ± 0.7, n = 7, 4 mo old, p < 0.0001). Furthermore, we show that DNT cells express significantly higher IL-17A than CD4+ cells after incubation with IL-23.
FIGURE 3 IL-23 induces IL-17A production in CD4+ and particularly in CD4−CD8− T cells. Cells were extracted from the lymph nodes of 5-mo-old B6, B6/lpr, 4-mo-old MRL/MPJ, and MRL/lpr mice. The cells were incubated in DMEM with plate-bound anti-CD3 (more ...)
Because IL-23 treatment led to increased Th17 expression among DNT in particular but also CD4+ cells, we asked whether IL-23 promotes T cell proliferation. T cells from MRL/lpr and control mice incubated with anti-CD3 Ab and IL-23 were cultured for 6 days. At the end of the incubation period, the cells were stained for CD4, CD3, and CD8. In , we show that the proportion of CD3+CD4−CD8− cells among T cells increased significantly (from 25 to 32.8% for MRL/MPJ mice; from 54 to 72.4% for MRL/lpr). Thereafter, we sorted CD3+CD4+ and CD3+CD4−CD8− cells and measured their proliferation. Two hundred thousand cells were incubated in 96-well plates precoated with anti-CD3 and anti-CD28 Abs in the absence or presence of IL-23 for 6 days. We found that DNT cells expanded significantly more in the presence of IL-23 (MRL/lpr DNT cells cultured with IL-23 vs MRL/lpr DNT cells cultured without IL-23: 600,000 ± 11,857 cells/well vs 320,000 ± 6,080 cells/well, p < 0.001; ). In contrast, IL-23 had no effect on CD4+ cells. From this set of experiments, we conclude that lupus-prone mouse T cells express high levels of IL-23 receptor mRNA and IL-17A mRNA and protein; incubation of the cells with IL-23 leads to a further significant increase in both IL-23 receptor and IL-17A expression. Finally, IL-23 acts as a trophic cytokine for DNT cells, increasing both their percentage among the total T cell population and their absolute number.
FIGURE 4 IL-23 stimulation expands the CD4−CD8− T cells derived from both control MRL/MPJ and lupus-prone MRL/lpr mice. A, Cells were extracted from the lymph nodes of 4-mo-old MRL/lpr and MRL/MPJ mice. The cells were incubated in DMEM with plate-bound (more ...)
IL-17-expressing cells are present in the kidneys of lupus-prone mice with nephritis
One of the most important manifestations of SLE is nephritis. A recent publication linked nephritis to the presence of IL-17A+
cells in the kidneys (5
). Since we found that T cells in the secondary lymphoid organs of lupus-prone mice express IL-17A at high levels, we evaluated whether similar T cell populations infiltrate the kidneys in these mice. shows immunohistochemical staining of kidneys from control and lupus-prone MRL/lpr
mice with active nephritis (5 mo old, protein in the urine >2000 mg/dl, RBC >2000 cells/dl urine, white blood cells >500 cells/dl urine). As can be readily seen, the kidneys contained a significant number of CD3+
cells in the tubulointerstitial area; we did not observe such cells in the glomeruli. In addition, we did not find such an infiltrate in the kidneys of control MRL/MPJ mice. Finally, CD3+
cells could be detected, albeit at smaller numbers, in the kidneys of 2-mo-old MRL/lpr
mice that did not show evidence of active nephritis (data not shown). Similar findings were also observed in B6/lpr
lupus-prone animals that suffer from a less severe form of nephritis (data not shown).
FIGURE 5 IL-17A- expressing T cells infiltrate the kidneys of lupus-prone mice. Frozen kidney sections from both 5-mo-old MRL/MPJ and MRL/lpr mice were stained with anti-CD3-FITC and anti-IL-17 Abs, followed by goat anti-rat IgG Texas Red fluorescent secondary (more ...)
IL-23-treated lymphocytes from lupus-prone mice induce nephritis in Rag-1−/− mice
Our data showed that IL-17A+ cells are present in both lymphoid organs and kidneys of lupus-prone mice with active nephritis. Our in vitro experiments established that IL-23 expands DNT, which is the main population of IL-17A+- expressing T cells in lupus-prone mice. To determine the role of IL-17A+ cells in inducing nephritis in lupus-prone mice, we conducted lymphocyte transfer experiments as follows: cells were harvested from the lymph nodes of lupus-prone B6/lpr and control B6 mice and activated them in vitro using an anti-CD3 Ab with or without IL23 for 48 h. At the end of the culture, 5 million cells were injected i.p. in mice that lack both B and T cells (Rag-1−/− mice). Before injecting the lymph node extracts in the recipient mice, we evaluated the expression of IL-17A+ in this cell population. As shown in , B6/lpr-derived cells had significantly more IL-17A+ cells compared with B6-derived cells. Furthermore there was a 2-fold increase in the number of IL-17A+ cells after treatment with IL-23 vs the non-IL-23-treated control group. In accordance with the above-mentioned experiments (see ), we found that the majority of cells producing IL-17A+ in these lymph node extracts are DNT cells (CD4+IL-17A+ cells from lymph node extracts of B6/lpr mice: 30.3 ± 1.05% of total cell population vs CD4−CD8−IL-17A+ cells: 67.4 ± 1.34% of total cell population, n = 4 animals). IL-23 treatment further increased the percentage of DNT cells in the IL-17A+ cell population (CD4+IL-17A+ cells from lymph node extracts of B6/lpr mice treated with IL-23: 19.6 ± 2.11% of total cell population vs CD4−CD8−IL-17A+ cells: 85.3 ± 0.83% of total cell population, n = 4 animals). Using CFSE staining, we evaluated the effect of IL-23 on proliferation of DNT cells. We found that IL-23 treatment increases the proliferation of DNT cells that are stimulated with anti-CD3 and anti-CD28 Abs for 4 days by 45% (dividing DNT cells stimulated with anti-CD3/anti-CD28 Abs for 4 days without IL-23: 34.4 ± 4.57% vs DNT cells stimulated with anti-CD3/anti-CD28 Abs for 4 days plus IL-23: 49.9 ± 5.16%). This finding suggests a “trophic” effect of IL-23 on DNT cells. Of note, <1% of the DNT cells were TCR γδ (as assessed by staining with anti-TCRγ Ab) and their percentage did not change with IL-23 treatment (data not shown).
FIGURE 6 IL-23 treatment leads to an increase in the number of IL- 17A+ lymphocytes and does not affect the percentage of B cells. Lymphocytes from 5- mo-old control B6 and lupus-prone B6/lpr mice were extracted (three mice in each group) and stimulated with anti-CD3 (more ...)
We also evaluated the effect of IL-23 treatment on CD19+ B cells. As shown in , treatment with IL-23 did not significantly change the percentage of B cells in lymph node extracts and therefore the absolute number of B cells injected in the recipient mice. Of note, B6/lpr mouse-derived lymph node extracts had ~2.5 times more B cells than control B6-derived lymph node extracts (percentage of B cells from B6/lpr-derived lymph node cells vs B6: 12.98% ± 1.69 vs 4.85 ± 0.79%, p = 0.022) (data not shown).
The Rag-1−/− mice that were transplanted with lymphocytes from B6 and B6/lpr mice were sacrificed 4 wk after transfer. First, we evaluated whether lymphocytes were engrafted in the secondary lymphoid organs. In , we show that CD3+ cells were found in Rag-1−/− spleens after the transplant. In addition (, fourth middle panel), mice that received B6/lpr-derived lymphocytes pretreated with IL-23 had CD3+IL-17A+ cells in their spleens. Very few CD3+IL-17A+ cells were seen in mice transplanted with non-IL-23-treated B6/lpr-derived lymphocytes. Finally, no IL-17A+ cells were seen in the spleens of Rag-1−/− mice transplanted with control B6-derived lymphocytes. This is in keeping with our observation that B6 and B6/lpr mice have very few resident IL-17A+ cells in their lymph nodes (0.32% vs 0.38%; see above).
FIGURE 7 IL-17A+CD3+ cells are found in the spleen of Rag-1−/− mice that were injected with IL-23-treated B6/lpr-derived lymphocytes. Rag-1−/− mice that were injected with lymphocytes derived from control B6 and B6/lpr mice were (more ...)
Subsequently, we evaluated whether CD3+ and in particular IL-17A+ cells could be found in the kidneys of the transplanted mice. Indeed, we found, similarly to the spleen, that only mice that received IL-23-treated B6/lpr-derived cells had IL-17A+ cells in their kidneys (). To further assess the presence of IL-17A+ cells in the cell infiltrate in kidneys of recipient mice, we extracted kidney cells from Rag-1−/− mice that were transplanted with B6-and B6/lpr-derived cells. These kidney cells were analyzed using flow cytometry. As shown in , flow cytometry confirmed that treatment of B6/lpr lymphocytes with IL-23 before their transfer in the recipient mice led to a 4-fold increase in IL-17A+ cells in the kidneys of recipient mice (percentage of IL- 17A+ cells in B6/lpr-derived IL-23-treated lymphocytes vs IL- 17A+ cells in B6/lpr-derived non-IL-23-treated lymphocytes: 0.049 ± 0.006 vs 0.014 ± 0.0007 p < 0.0001; n = 4). As expected, mice that received B6-derived lymphocytes did not have IL-17A+ cells in their kidneys. Importantly, IL-17A+ cells in the kidneys of recipient mice were mainly CD4−CD8− cells. Since these cells were treated with IL-23 before the transfer, this finding was in keeping with our in vitro experiments showing a trophic effect of IL-23 on CD4−CD8− cells.
FIGURE 8 IL-17A+CD3+ cells are found in kidneys of Rag-1−/− mice that were injected with IL-23-treated B6/lpr-derived lymphocytes. Rag-1−/− mice that were injected with lymphocytes derived from control B6 and B6/lprmice were sacrificed (more ...)
To address whether IL-17A+ infiltration of kidneys in recipient Rag-1−/− mice leads to overt nephritis, we evaluated the urine of the recipient mice for the presence of inflammatory cells (white blood cells) and protein. As shown in , transfer of IL-23-treated lymphocytes in Rag-1−/− mice led to significant above the baseline proteinuria and appearance of white blood cells in the urine. The pyuria developed as early as 3 wk after transplant and disappeared soon afterward (at 4 wk). The proteinuria appeared shortly after the pyuria (by 4 wk), persisted for 4 wk, and gradually disappeared at 12 wk after transplant. Mice that were transplanted with B6-derived lymphocytes and B6/lpr lymphocytes that were not treated with IL-23 did not show evidence of significant proteinuria or pyuria.
Urine analysis and kidney pathology of Rag 1−/−recipient mice that were injected with lymph node extracts from control and lupus-prone mice
Ig and C3d deposition is increased in the kidneys of Rag-1−/− mice transplanted with IL-23-treated lupus-prone mouse-derived lymphocytes
Lupus nephritis is characterized by the invariable deposition of Ig (IgG, IgM, IgA) and C3 in the glomeruli (4
). We therefore evaluated the kidneys of the recipient mice for the presence of IgG, IgM, and C3d. In , we show that Rag-1−/−
mice that received lupus-prone mouse-derived lymphocytes had deposition of Ig (both IgG and IgM) as well as C3d in their glomeruli. This was much more apparent in mice that received lymphocytes treated with IL-23 (, row 4
). No Ig or complement deposition was seen in kidneys of Rag-1−/−
mice that received control B6-derived lymphocytes (, rows 1
FIGURE 9 Rag-1−/− mice injected with IL-23-treated B6/lpr-derived lymphocytes show significant IgG, IgM, and C3 deposition in the kidneys. Rag-1−/− mice that were injected with lymphocytes derived from control B6 and B6/lpr mice (more ...)
Subsequently, we stained the kidneys of the recipient mice with H&E and PAS. shows that Rag-1−/− mice that were transplanted with IL-23-treated lupus-prone mouse-derived lymphocytes had obliteration of the urine space of the glomerulus, mesangial expansion, and endocapillary cell proliferation. They also exhibited mild basement membrane thickening (PAS stain) and had rare interstitial cell infiltrates (photographs not shown). In addition, we measured the average glomerular area (according to the technique described in Materials and Methods). We found () that transfer of IL-23-treated lymphocytes derived from lupus-prone mice, but not control mice, led to ~25% increase in the average glomerular area. In addition, we found that there was a nonstatistically significant increase in the average number of cells in the glomeruli of animals that were injected with B6/lpr-derived lymph node cells vs B6-derived lymph node cells; pre-treatment of the B6/lpr lymph node extracts with IL-23 resulted in even higher numbers of glomerular cells in the recipient mice ().
FIGURE 10 Rag-1−/− mice injected with IL-23-treated B6/lpr-derived lymphocytes have enlarged glomeruli in their kidneys. Rag-1−/− mice that were injected with lymphocytes derived from control B6 and B6/lpr mice were sacrificed 4 (more ...)