IL-23R deficiency prevents hyperplasia of secondary lymphoid organs in lupus-prone mice
We have shown (3
) that when B6/lpr
-derived lymphocytes are cultured in the presence of IL-23, they acquire the capacity to become pathogenic. Accordingly, we hypothesized that IL-23–mediated signaling is central in the pathogenesis of lupus. To address this question, we generated lupus-prone B6/lpr
mice lacking IL-23R. These IL-23R−/−
mice were healthy and fertile. We observed the mice for a period of 9 mo for the development of signs of lupus and lupus nephritis. We found that the IL-23R−/−
mice developed milder proteinuria than age- and sex-matched B6/lpr
wild-type mice; IL-23R−/−
mice were similar to control B6 mice in terms of proteinuria (IL-23R−/−
versus B6, protein in urine: 100 mg/dl versus 2000 mg/dl versus 100 mg/dl; n
= 5). More importantly, there was no evidence of pyuria in the IL-23R−/−
mice as opposed to the B6lpr
We then sacrificed 9-mo-old B6/lpr, IL-23R−/−B6/lpr, and B6 mice. Although at this age B6/lpr mice exhibit severe disease manifestations including hyperplasia of the secondary lymphoid organs and glomerulonephritis, mice that lacked IL-23R had significantly smaller spleens and lymph nodes than wildtype lupus-prone mice (). The size of the lymphoid organs approximated those of a normal B6 mouse (not shown in this paper). The reduced size of the lymphoid organs in the IL-23R−/−B6/lpr mice was associated with a profound reduction in the cell population in these organs by ~50% in the spleen and 90% in the lymph nodes [B6/lpr (n = 5) versus IL-23R−/−B6/lpr (n = 5) versus B6 (n = 4) total splenocytes × 106 ± SD: 186.4 ± 8.5 versus 112.26 ± 2.58 versus 124.15 ± 5.08; p = 0.01; B6/lpr (n = 5) versus IL-23R−/−B6/lpr (n = 5) versus B6 (n = 4) total lymph node cells × 106 ± SD: 672.3 ± 35.5 versus 66 ± 9.17 versus 40 ± 8.27; p = 0.016; ).
FIGURE 1 IL-23R−/−B6/lpr mice have smaller secondary lymphoid organs and significantly reduced number of CD3+ and CD3+CD4−CD8− cells as compared with B6/lpr mice. A, The secondary lymphoid organs were harvested from 9-mo-old B6/ (more ...)
In evaluating the subsets of lymphocytes in the IL-23R−/−
mice, we found that the total number ofCD3+
T cells in both spleens and lymph nodes are reduced when compared with wildtype B6/lpr
= 5) versus IL-23R−/−
= 5) versus B6 (n
splenocytes × 106
± SD: 36.42 ± 3.5 versus 27.41 ± 3.8 versus 28.44 ± 1.4; p
= 0.04 for B6/lpr
=5) versus IL-23R−/−
=5) versus B6 (n
lymph node cells × 106
± SD: 338.49 ± 21.4 versus 26.07 ± 1.12 versus 12.6 ± 0.45; p
= 0.04 for B6/lpr
; ]. In analyzing T cell subsets, we found that the IL-23R−/−
mice had a significant decrease in the number of CD3+
(double-negative T cells [DNTs]) that accumulate abnormally in the lymphoid organs of lupus-prone mice that bear the lpr
= 5) versus IL-23R−/−
= 5) versus B6 (n
= 4) DNT percent ofCD3+
splenocytes: 24.5 ± 2.4 versus 17 ± 1.78 versus 6.73 ± 1.04; p
= 0.025 for B6/lpr
; DNT percent among CD3+
lymph node cells: 32.89 ± 2.78 versus 9.87 ± 1.19 versus 6.8 ± 0.84; p
= 0.0002 for B6/lpr
). A representative experiment is shown in and cumulative data in . Conversely, we observed a significant rise in the percentage of CD8 cells in both spleen (p
= 0.0079) and lymph nodes (p
= 0.0317) of IL-23R−/−
mice (). We did not observe significant changes in the percentage of CD4+
cells between the two groups (). It has to be stated, although, that because the overall number of lymphocytes in the lymph nodes of the IL-23R– deficient mice was profoundly reduced, the overall number of DNT, CD4, and CD8 T cells in the lymph nodes was significantly decreased (). The percentage of regulatory T (CD4+
) (Supplemental Fig. 1
), γδ T, B, dendritic, and monocyte lineage cells did not differ significantly between the IL-23R−/−
mice. Additionally, there was no difference in T cell subtypes, such as DNT and regulatory T cells, between B6 and IL-23R−/−
B6 (Supplemental Fig. 2
This set of experiments shows that in the absence of IL-23R, the accumulation of lymphocytes and the generation of the abnormal double-negative cells are profoundly reduced.
IL-23R deficiency resulted in reduced production of inflammatory T cell-derived cytokines and autoantibodies
It has been shown that the development of lupus nephritis in mice depends on the production of proinflammatory cytokines, such as IFN-γ (9
) and, more recently, IL-17. Because IL-23R deficiency resulted in profound changes in the T cell phenotype in lupus-prone mice, we hypothesized that the production of proinflammatory cytokines and, in particular, IL-17A was impaired in the IL-23R−/−
Indeed, as shown in , the overall number of cells producing IL-17A is profoundly decreased in the IL-23R−/−B6/lpr mice (B6/lpr versus IL-23R−/−B6/lpr CD3+IL-17+ lymph node cells ± SD: 813,800 ± 204,800 versus 8,020 ± 1,880; n = 5; , lower panel). Both CD4+IL-17+ and CD3+CD4−CD8−IL-17+ cells were decreased in the IL-23R−/−B6/lpr mice versus B6/lpr mice. Additionally, the percentage of DNTs expressing IL-17+ was significantly decreased in IL-23R−/−B6/lpr mice (, right panel). In the CD4+ population, there were no significant differences between the two mice in percentage of IL-17+ cells.
FIGURE 2 IL-23R−/−B6/lpr mice have decreased number of IL-17+ cells, serum IgG, and anti-dsDNA Ab levels as compared with B6/lpr mice. A, Lymph node-derived cells from both IL-23R−/−B6/lpr and B6/lpr mice were stained for intracellular (more ...)
Because development of lupus depends on IFN-γ, we evaluated expression of this cytokine by both CD4+
and DNTs. As shown in , both CD4+
and DNTs from IL-23R−/−
mice had reduced expression of IFN-γ when compared with control B6/lpr
mice. These findings suggest not only that DNTs (and to a lesser extent CD4+
cells) are reduced in number in IL-23R−/−
mice, but also that the production of proinflammatory cytokines is significantly decreased. When comparing B6 to IL-23R−/−
B6 mice, there was a nonstatistically significant decrease of the expression of IFN-γ and IL-17A by DNTs in the IL-23R–deficient mice (Supplemental Fig. 3
). It has to be stated, although, that both cytokines were expressed at very low levels by DNTs in both strains.
We have shown in the past that IL-23 treatment of B6/lpr-derived lymphocytes that were later transferred to Rag-1−/− mice results in IgG deposition in the kidneys of recipient mice. We asked the question whether abrogation of the IL-23–mediated signaling would affect the production of Ig and, more importantly, of pathogenic autoantibodies. Indeed, as shown in , the levels of IgG in the serum of IL-23R−/−B6/lpr mice is significantly reduced when compared with control B6/lpr mice [B6/lpr (n = 8) versus IL-23R−/−B6/lpr (n = 7) versus B6 (n = 3) total serum IgG (ng/ml): 394418.75 ± 28155.85 versus 182571.42 ± 11330.09 versus 135250 ± 19440.99; p = 0.0002 for B6/lpr versus IL-23R−/−B6/lpr]. More importantly, the IL-23R–deficient mice produced less anti-dsDNA Abs than B6/lpr mice and at similar levels to normal B6 mice [B6/lpr (n = 9) versus IL-23R−/−B6/lpr (n = 10) versus B6 (n = 4) serum anti-dsDNA Ab levels (IU/ml): 3424.44 ± 481.31 versus 1365.29 ± 143.52 versus 836.23 ± 75.54; p = 0.0003 for B6/lpr versus IL-23R−/−B6/lpr; ].
The above findings point to the fact that in the absence of IL-23R, lupus-prone mice do not exhibit some of the readily recognizable features of immune dysregulation characteristic of lupus.
IL-23R deficiency mitigates glomerulonephritis in lupus-prone mice
As shown above, IL-23R−/−B6/lpr mice are characterized by significantly decreased spontaneous expression of inflammatory cytokines and production of autoantibodies. Therefore, we evaluated whether glomerular and interstitial inflammation that characterizes B6/lpr lupus-prone mice is decreased in the IL-23R−/−B6/lpr mice. As shown in , glomeruli from IL-23R−/−B6/lpr mice displayed reduced mesangial cell proliferation and membrane thickening when compared with B6/lpr [glomerular damage grade: B6/lpr (n = 8) versus IL-23R−/−B6/lpr (n = 7) versus B6 (n = 4): 2.2 ± 0.14 versus 0.8 ± 0.14 versus 0.7 ± 0.13; p = 0.0001 for B6/lpr versus IL-23R−/−B6/lpr]. In parallel, the IL-23R−/−B6/lpr mice had decreased perivascular infiltration with lymphocytes when compared with B6/lpr [B6/lpr (n = 8) versus IL-23R−/−B6/lpr (n=7) versusB6(n = 4): 1.86 ± 0.31 versus 0.57 ± 0.28 versus 0.75 ± 0.5; p = 0.02 for B6/lpr versus IL-23R−/−B6/lpr; ]. Although all B6/lpr kidneys had perivascular infiltration with inflammatory cells, only one out of seven IL-23R−/−B6/lpr mice and one out of four B6 mice had evidence of perivascular inflammation in the kidneys.
FIGURE 3 IL-23R−/−B6/lpr mice do not develop significant nephritis. Nine-month-old IL-23R−/−B6/lpr, B6/lpr, and B6 mice were sacrificed and their kidneys harvested and stained. A, H&E and periodic acid-Schiff staining of (more ...)
Because we found that the IL-23R−/−B6/lpr mice had also decreased Ig and anti-dsDNA Ab levels in the serum when compared with B6/lpr, we stained the glomeruli for IgG, IgM, and C3 deposition. As shown in , the intensity of the staining for both was significantly reduced in the IL-23R– deficient mice-derived kidneys when compared with B6/lpr. We also stained the kidneys for IL-17A and found rare IL-17+ cells infiltrating the kidneys of B6/lpr mice but not the kidneys of IL-23R−/−B6/lpr mice (data not shown).
Given these findings in IL23R−/−
mice, we used an Ab against IL-23 to treat 12-wk-old MRL/lpr
mice for 4 wk. We found that at the end of the treatment, the anti–IL-23 Ab-injected mice had decreased proteinuria compared with IgG control-treated mice (100 mg/dl versus 2000 mg/dl; n
= 3 for each group). The anti–IL-23 Ab-treated mice did not have any hematuria or pyuria, whereas the control IgG-treated mice had both (Z. Zhang, V.C. Kyttaris, and G.C. Tsokos, unpublished observation). Moreover, IL-17A production was significantly decreased in the anti–IL-23–treated mice as compared with controls (Supplemental Fig. 4
Our previous studies showed that treatment with IL-23– pretreated B6/lpr lymph node-derived cells were capable of causing glomerulonephritis when transferred to Rag-1−/− mice. This study expands our previous understanding of the contribution of IL-23 to the pathogenesis of lupus by showing for the first time that IL-23R is necessary for lymphoid hyperplasia, production of pathogenic autoantibodies, and infiltration of kidneys by IL-17+ cells in lupus-prone mice. Furthermore, the spontaneous secretion of IFN-γ is decreased in the IL-23R−/− lupus-prone animals as a consequence of global decrease in spontaneous immune system activation. These findings coupled with the fact that proteinuria was decreased and pyuria was not present in the IL-23R–deficient mice point to the fact that IL-23R represents a key molecule in the chain reaction that leads to lupus nephritis. This conclusion is further reinforced by our preliminary data on treatment of lupus-prone mice with an anti–IL-23R Ab showing reversal of the proinflammatory phenotype and clinical improvement of these mice.
The most striking immunologic feature of the IL-23R−/−
mouse was the significant decrease of DNTs, the major source of IL-17A in lupus-prone mice. Our data strongly suggest that these cells may act as effector cells (10
), orchestrating the inflammatory response in the kidneys and/or providing help to B cells to produce pathogenic autoantibodies (11
). The extent to which each of these pathways contributes to the development of lupus nephritis remains to be determined. Additional studies will also be needed to further elucidate the exact cytokines secreted by Th17 cells that contribute to lupus pathology.
In summary, this study demonstrates that IL-23R deficiency limits the lymphoid hyperplasia, abrogates the accumulation of DNTs, and prevents the development of nephritis in lupus-prone mice. Inhibition of IL-23 and/or IL-17A in vivo at various stages of lupus will enable us to determine the optimal time to block this pathway to prevent lupus nephritis.