The uninflamed pancreas harbors cDCs that express high levels of TLR3. As alluded to in the Introduction, the model of AIP/IgG4-RD in MRL/MpJ mice induced by repeated (16-fold) administration of poly(I:C) is mainly driven by activated pDCs (3–5). This introduces a conundrum because in previous studies, while pDCs isolated from uninflamed splenic tissue express high levels of TLR7 and TLR9, they lack expression of the TLR capable of responding to poly(I:C), TLR3; on the other hand, in the same studies, isolated CD11c+ cDCs were found to express high levels of TLR3 (8). To determine whether a similar situation exists in uninflamed pancreatic tissue of MRL/MpJ mice, pDCs and cDCs in pancreatic mononuclear cells (PMNCs) from these mice were purified by flow cytometric sorting on the basis of PDCA-1hiB220lo and CD11chiMHC class IIhi surface markers, respectively, and then analyzed by quantitative reverse transcription PCR (qPCR) to determine mRNA expression of Tlr3 and Tlr9 (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.167910DS1). We found that whereas both pancreatic cDCs and pDCs express higher levels of Tlr3 mRNA than counterpart cells in the spleen, cDCs are by far the main TLR3-expressing cells in uninflamed pancreatic tissue (Supplemental Figure 1B). In addition, both splenic and pancreatic pDCs from uninflamed tissues expressed only modestly higher levels of Tlr9 mRNA than splenic cDCs (Supplemental Figure 1B). These results indicate that cDCs are more likely than pDCs to be the major cell initiating murine AIP upon repeated administration of poly(I:C).

Blockade of TLR3 inhibits the development of experimental AIP. We next determined whether the development of experimental AIP requires recognition of poly(I:C) by TLR3-expressing cells in the uninflamed pancreas. To this end, we performed TLR3 inhibition studies utilizing a well-studied competitive inhibitor of double-stranded RNA (dsRNA) binding to TLR3 [(R)-2-(3-chloro-6-fluorobenzo(b)thiophene-2-carboxamido)-3-phenylpropanoic acid] to determine whether inhibition of TLR3 signaling blocks development of AIP; importantly, this inhibitor has previously been shown not to inhibit signaling by other TLRs, including signaling by TLR7 (18, 19).

We found that whereas repeated administration of both poly(I:C) and saline control to MRL/MpJ mice led, as expected, to the development of AIP characterized histologically by destruction of pancreatic acinar architecture, infiltration of immune cells, and massive fibrosis, repeated injections of both poly(I:C) and the inhibitor of TLR3 signaling led to only minor histologic changes of this kind (Figure 1, A and B). By flow cytometric analysis of PMNCs from mice subjected to TLR3 blockade and control mice (Supplemental Figure 2A), we also found that blockade of TLR3 signaling led to a great reduction in the cellular immune response previously shown to cause AIP, i.e., a reduction in the pancreatic accumulation of pDCs (defined as PDCA-1+B220lo cells) and CD3+ T cells (Figure 1C). In addition, it led to reduction in the accumulation of CD11b+ myeloid cells and CD11c+ DCs (Figure 1C) and to a reduction in their CD11b and CD11c mean fluorescence intensities (MFIs) (Supplemental Figure 2B). Likewise, blockade of TLR3 signaling was accompanied by decreased pancreatic expression of IFN-α and IL-33, both of which have been shown to be produced by infiltrating pDCs in this model of AIP (3–6) (Figure 1D). Finally, blockade led to decreased pancreatic expression of CXCL9 and CXCL10, Th1 chemokines dependent on type I IFN responses (Figure 1D) (20).

Blockade of TLR3 by inhibitors prevents the development of experimental autoimmune pancreatitis. MRL/MpJ mice were administered poly(I:C) by intraperitoneal injection twice a week for a total of 16 times to induce experimental autoimmune pancreatitis (AIP). Each poly(I:C) injection was preceded by intraperitoneal injection of saline (PBS, n = 4) or TLR3/dsRNA binding inhibitor (1 mg, n = 4). After sacrifice at 3 hours following the final set of injections, pancreases were removed and analyzed as indicated. (A and B) Hematoxylin and eosin staining of the pancreatic tissues and pathological scores of induced AIP in the 2 groups. Original magnification, ×400. (C) Percentages of plasmacytoid DCs (pDCs), CD3+ T cells, CD11b+ myeloid cells, and CD11c+ DCs within pancreatic mononuclear cells, as determined by flow cytometric analyses. pDCs were defined as PDCA-1+B220lo cells. Left panels: Representative cytometric analysis. Right panels: Bar graphs of cumulative results. (D) Concentrations of IFN-α, IL-33, CXCL9, and CXCL10 in protein extracts of pancreatic tissues from mice without and with TLR3 inhibitor administration, as determined by ELISA. Each dot corresponds to the value in 1 mouse. Statistical analyses were performed using an unpaired, 2-tailed Student’s t test. Results are expressed as mean ± SEM. **P < 0.01.

The above data strongly suggested that resident cDCs bearing TLR3 were the cells responding to poly(I:C) during the early induction phase of AIP. To verify that this was the case, we determined the responses of purified pancreatic cDCs and pDCs to poly(I:C) isolated from mice in the early phase of AIP induction (i.e., after administration of a second dose of poly[I:C]). We found that at this early time point the cDCs, but not the pDCs, produced large amounts of IFN-α, IFN-β, CXCL9, and CXCL10 upon stimulation with poly(I:C) (Figure 2A); pDCs could also produce these factors, but only when stimulated by CpG, a TLR9 ligand that was not the initiating driver of disease. Finally, it should be noted that the cDCs isolated from poly(I:C)-treated mice produced higher levels of type I IFNs and chemokines upon stimulation with poly(I:C) than cDCs from untreated mice, suggesting that cDCs present in the milieu characterizing the early phase of AIP have gained enhanced responsivity to TLR3 stimulation.

Conventional DCs are the major cellular source of type I IFNs in the inductive phase of experimental autoimmune pancreatitis. MRL/MpJ mice (n = 6) were treated with poly(I:C) by intraperitoneal injection for a total of 2 times. After sacrifice at 3 hours following the final injection, pancreases were removed and processed to extract pancreatic mononuclear cells (PMNCs). PMNCs were extracted from untreated MRL/MpJ mice (n = 6) to obtain baseline data (No Tx). (A) Conventional DCs (cDCs, 1 × 106/mL) and pDCs (1 × 106/mL) were purified from the extracted PMNCs and cultured with poly(I:C) (25 μg/mL) or CpG (1 μM) for 48 hours in triplicate. Culture supernatants from each well were then analyzed by ELISA to determine the concentrations of IFN-α, IFN-β, CXCL9, and CXCL10. (B) PMNCs (1 × 106/mL) and CD11c+ DC–depleted PMNCs (1 × 106/mL) were stimulated with poly(I:C) (25 μg/mL) for 48 hours in triplicate. Culture supernatant from each well was then analyzed by ELISA. Each dot represents the value derived from 1 well. Statistical analyses were performed using an unpaired, 2-tailed Student’s t test. Results shown are representative of data derived from 2 independent experiments and are expressed as mean ± SEM. **P < 0.01.

TLR3 expression is not limited to the CD11c+ cDCs (21). Therefore, in a final set of studies relating to TLR3 expression in early experimental AIP, we performed cell depletion studies to verify that cDCs were indeed the main source of type I IFNs at this stage of disease. Accordingly, PMNCs isolated from the mice after treatment with the second dose of poly(I:C) were depleted of CD11c+ cells by anti-CD11c MACS beads and then stimulated with poly(I:C). As previously observed, PMNCs isolated from poly(I:C)-treated mice produced greater amounts of IFN-α and IFN-β than those from untreated mice (Figure 2B); however, depletion of CD11c+ cells markedly reduced production of type I IFNs. Thus, pancreatic cDCs, at this early phase of AIP induction, are indeed the main producers of type I IFNs upon sensing of poly(I:C) by TLR3 and the possible contribution of other cell types to such production is small.

Taken together, these studies provide evidence that the development of experimental AIP following repeated poly(I:C) administration is initiated by TLR3+ cDCs producing type I IFNs, CXCL9, and CXCL10.

The MRL/MpJ model of experimental AIP is marked by migration of CXCR3+ T cells into the pancreas. Recognizing that poly(I:C) activation of TLR3 in cDCs residing in the uninflamed pancreas and then in the nascent inflamed pancreas would induce production of type I IFNs and Th1 chemokines (CXCL9, CXCL10), we reasoned that poly(I:C) induction of AIP could be accompanied by an early downstream Th1 cell–oriented cytokine and chemokine response that could lead to induction of Th1 cells in lymph nodes draining the pancreas and their migration into the pancreas. In studies addressing this possibility, we found that the percentage of pancreatic CD4+CXCR3+ T cells (i.e., Th1 cells) among PMNCs in MRL/MpJ mice administered poly(I:C) 16 times was much greater than those of untreated mice, whereas in contrast, such treatment did not increase the percentage of CD8+CXCR3+ T cells (Figure 3A) and caused a slight decrease in the percentage of CCR4+ cells within the CD3+ T cell population (i.e., Th2 cells) (Supplemental Figure 2C). As expected from the presence of CXCR3+ Th1 cells, we found that the levels of IFN-γ and TNF-α were greater in the pancreas of poly(I:C)-treated mice than in the pancreas of untreated mice, whereas no significant difference was seen in the levels of IL-4 or IL-17 in these groups of mice (Figure 3B). In addition, pancreatic expression of CXCL9 and CXCL10 (i.e., chemokines targeting CXCR3 on Th1 cells; ref. 20) was markedly enhanced in treated mice as compared with those in untreated mice, whereas in contrast, pancreatic expression of CCL17 and CCL22, chemokines targeting CCR4 expressed on Th2 cells (20), was comparable in mice with and without poly(I:C) treatment (Figure 3C). Finally, since CD4+ cytotoxic T lymphocytes (CTLs) expressing SLAMF7, granzyme A, or granzyme B are implicated in the immunopathogenesis of human IgG4-RD (9, 17), we determined whether the CD4+CXCR3+ T cells isolated from the pancreas of mice administered poly(I:C) express SLAMF7 or granzyme B by cell surface or intracellular flow cytometric staining, respectively, but found no such staining (Supplemental Figure 3A). Thus, it is unlikely that CD4+ CTLs play a major role in the pathogenesis of experimental AIP at this stage of the disease.

Expression of Th1-associated cytokines and chemokines is enhanced in MRL/MpJ mice treated with repeated injections of poly(I:C). MRL/MpJ mice (n = 14) were treated with poly(I:C) by intraperitoneal injection twice a week for a total of 16 times. After sacrifice at 3 hours following the final injection, pancreases were removed and processed to extract pancreatic mononuclear cells (PMNCs) and proteins. PMNCs and proteins were extracted from untreated MRL/MpJ mice (n = 10, No Tx) to obtain baseline data. (A) Representative flow cytometric analyses (with gates set on lymphocyte fraction, upper panel) and bar graphs of cumulative results (lower panel) showing the percentages of CD4+CXCR3+ T cells and CD8+CXCR3+ T cells among PMNCs obtained from individual mice. (B and C) Concentrations of IFN-γ, TNF-α, IL-4, IL-17, CXCL9, CXCL10, CCL17, and CCL22 in protein extracts of pancreatic tissues from mice. Each dot represents the value derived from 1 mouse. Statistical analyses were performed using an unpaired, 2-tailed Student’s t test. Results shown represent the combined data of 3 (A) or 2 (B) independent experiments. Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01.

The above characterization of pancreatic CD4+CXCR3+ T cells was performed on PMNCs isolated from MRL/MpJ mice after a full poly(I:C) 16-dose administration regimen when the pancreas was already heavily populated by pDCs. It was therefore of interest to determine whether a similar T cell population was present in the pancreas at an earlier time point when, as shown above, pancreatic cDCs rather than pDCs were still the main source of type I IFNs and chemokines attracting CD4+CXCR3+ T cells into the pancreas. To explore this question, we compared accumulation of CD4+CXCR3+ T cells as assessed by percentage of CD4+CXCR3+ T cells among PMNCs after administration of 3 doses of poly(I:C) with that after administration of 16 doses of poly(I:C). We found that whereas the percentage of pDCs was only mildly increased after 3 doses, the percentage of CD4+CXCR3+ T cells after 3 doses was equal to that after 16 doses (Supplemental Figure 3B). This early accumulation of CD4+CXCR3+ T cells is fully consistent with our hypothesis that initial stimulation of pancreatic TLR3-bearing cDCs by poly(I:C) results in IFN-α/β production and that the latter, in turn, leads to induction of Th1 responses (most likely in lymph nodes draining the pancreas) followed by early migration of CXCR3+ T cells into the pancreas via CXCL9 and CXCL10 secretion.

The development of experimental AIP depends on the interaction between CXCL9 or CXCL10 and CXCR3. The above data showing that CXCL9 and/or CXCL10 expression was associated with accumulation of CXCR3+ T cells in the inflamed pancreas of mice with experimental AIP raised the question of whether this CXCL9/CXCL10-CXCR3 interaction is required for the development of AIP. To answer this question, we determined the effect of blocking CXCL9/CXCL10-CXCR3 interaction on development of AIP by administration of a neutralizing antibody (Ab) against CXCR3 or control Ab at the time of each poly(I:C) injection. We found that, as first evaluated by AIP scores, such blockade prevented the development of experimental AIP (Figure 4, A and B). Moreover, this attenuation of AIP was accompanied by a reduction in the pancreatic accumulation of pDCs as well as CD3+ T cells; in addition, it was accompanied by the reduced percentages of CD11b+ myeloid cells and CD11c+ DCs with reduced CD11b and CD11c expression, as determined by MFI (Figure 4C and Supplemental Figure 2B). Finally, consistent with the reduction in pancreatic pDCs, pancreatic expression of IFN-α, IL-33, and IFN-γ was also markedly decreased in the anti-CXCR3 Ab plus poly(I:C)–treated mice as compared with those treated with control Ab plus poly(I:C) (Figure 4D). These data strongly suggest that the chemoattraction and accompanying migration of CXCR3+ T cells into the pancreas mediated by CXCL9 and/or CXCL10 is necessary for the development of experimental AIP. In addition, since such migration was required for proinflammatory cytokine production, these data also imply that the interaction between CD4+CXCR3+ T cells and CD11c+ DCs during the induction phase of AIP is indispensable for the subsequent development of mature AIP. Parenthetically, the dependence of the pancreatitis on these chemokine ligand-receptor interactions also implies that Th1 cell development is occurring outside of the pancreas.

The development of experimental autoimmune pancreatitis is dependent on the CXCR3-mediated signaling pathways. MRL/MpJ mice were administered poly(I:C) twice a week for a total of 16 times by intraperitoneal injection. Each poly(I:C) injection was preceded by intraperitoneal injection of anti-CXCR3 Ab (200 μg, n = 7) or control Ab (200 μg, n = 9). After sacrifice at 3 hours following the final set of injections, pancreases were removed and analyzed as indicated. (A and B) Representative hematoxylin and eosin staining of the pancreatic tissues and pathological scores for autoimmune pancreatitis of individual mice in the 2 groups. Original magnification, ×400. (C) Flow cytometric analyses showing the percentages of pDCs, CD3+ T cells, CD11b+ myeloid cells, and CD11c+ DCs among pancreatic mononuclear cells. pDCs were defined as PDCA-1+B220lo cells. Left panels: Representative flow cytometric analyses. Right panels: Bar graphs of cumulative data from individual mice. (D) Concentrations of IFN-α, IL-33, and IFN-γ in protein extracts of pancreatic tissues obtained from mice as determined by ELISA. Statistical analyses were performed using an unpaired, 2-tailed Student’s t test. The results shown are the combined data derived from 2 independent experiments. Each dot represents the value derived from 1 mouse. Results are expressed as mean ± SEM. **P < 0.01.

Pancreatic accumulation of pDCs requires interaction between CCR9 and CCL25. As indicated above, the initial source of type I IFNs mediating pancreatic migration of CXCR3+ T cells is likely to be TLR3-bearing pancreatic cDCs susceptible to stimulation by poly(I:C) rather than pancreatic pDCs, since the latter cells express low levels of TLR3.

However, the development of AIP/IgG4-RD is accompanied by a huge increase in the number of pancreatic pDCs and the latter cells (not cDCs) are the source of IFN-α in the mature inflammation. Indeed, the kinetic studies described above clearly showed that the percentages of pancreatic pDCs were much higher in mice treated with 16 doses of poly(I:C) than in those treated with zero or 3 doses (Supplemental Figure 3B). This raised the question of the mechanism underlying pDC accumulation in the nascent inflamed pancreas. Accordingly, we next investigated the chemokine interactions that could facilitate pDC migration into the pancreas in the next stage of developing experimental AIP.

In initial studies, we determined that pancreatic pDCs isolated from mice with poly(I:C)-induced AIP, as do pDCs generally, express CCR2, CCR7, and CCR9 (22) (Figure 5A). We therefore next assessed pancreatic expression of chemokine ligands that bind to and attract pDCs bearing these receptors, CCL2, CCL21, and CCL25 (23–25). We found that following administration of repeated doses of poly(I:C), pancreatic tissue expressed increased levels of CCL2 and CCL25, but not CCL21 (Figure 5B), suggesting that pDCs utilize molecular interactions between CCR2 and CCL2 or between CCR9 and CCL25 to migrate to the pancreas.

Expression of CCR9 on pDCs in MRL/MpJ mice treated with repeated injections of poly(I:C). MRL/MpJ mice were treated with or without poly(I:C), as indicated in Figure 3. After sacrifice at 3 hours following the final set of injections, pancreases were removed and analyzed as indicated. (A) Flow cytometric analysis of cell membrane expression of CCR2, CCR7, and CCR9 on pDCs in pancreatic mononuclear cells extracted from MRL/MpJ mice treated with 16 doses of poly(I:C). Analysis gate was set on PDCA-1+B220lo pDCs. Black curve: control Ab. Red curve: CCR2, CCR7, and CCR9 Abs. Results shown are representative data from 3 mice. (B) Concentrations of CCL2, CCL21, and CCL25 in protein extracts of pancreatic tissues obtained from untreated mice (No Tx, n = 10) or mice treated with 16 injections of poly(I:C) (n = 10), as determined by ELISA. Each dot represents the value derived from 1 mouse. Statistical analyses were performed using an unpaired, 2-tailed Student’s t test. Results shown are combined data of 2 independent experiments (B). Results are expressed as mean ± SEM. **P < 0.01.

To determine whether these interactions were in fact necessary for the development of pancreatitis, we first assessed the effect of neutralizing Ab against CCL2 or CCL25 administered at the time of poly(I:C) administration. We found that the development of AIP, as measured by pathological scores, was comparable between mice treated with control Ab or with anti-CCL2 Ab (Figure 6, A and B). Likewise, administration of this Ab was not accompanied by significant changes in pancreatic expression of IFN-α, IL-33, or TNF-α (Figure 6C). Finally, administration of anti-CCL2 Ab did not reduce the percentage of pDCs as compared with administration of control Ab, although such treatment reduced the percentages of CD11b+ myeloid cells and CD11c+ DCs and tended to reduce their expression of CD11b and CD11c (Figure 6D and Supplemental Figure 2B). It should be noted that since the reduction in CD11c+ DC percentage by anti-CCL2 Ab administration is likely to be a consequence of cell migration, it reflects the lack of endogenous expansion of the initially present resident CD11c+ DC population during the development of pancreatitis; thus, the reduction in CD11c+ DCs suggests that this cell population is dispensable when the pancreatic inflammation has reached a mature stage.

Pancreatic accumulation of pDCs does not require the molecular interaction between CCR2 and CCL2. MRL/MpJ mice were administered poly(I:C) twice a week for a total of 16 times. Each poly(I:C) injection was preceded by intraperitoneal injection of anti-CCL2 Ab (200 μg, n = 5) or control Ab (200 μg, n = 5). After sacrifice at 3 hours following the final set of injections, pancreases were removed and analyzed as indicated. (A and B) Representative hematoxylin and eosin staining of the pancreatic tissues and pathological scores of induced autoimmune pancreatitis in the 2 groups. Original magnification, ×400. (C) Concentrations of IFN-α, IL-33, TNF-α, and IL-6 in protein extracts of pancreatic tissues obtained from mice in the 2 groups. (D) Flow cytometric analyses showing the percentages of pDCs, CD3+ T cells, CD11b+ myeloid cells, and CD11c+ DCs among pancreatic mononuclear cells. Left panels: Representative flow cytometric analyses. Right panels: Bar graphs of cumulative data from individual mice. pDCs were defined as PDCA-1+B220lo cells. Each dot represents the value derived from 1 mouse. Statistical analyses were performed using an unpaired, 2-tailed Student’s t test. Results are expressed as mean ± SEM. **P < 0.01.

In parallel studies, we investigated the involvement of the interaction between CCR9 and CCL25 by assessment of pancreatitis following administration of anti-CCL25 Ab or control Ab. In this case, we found that, as determined by pathological scores, the blockade of the CCR9-CCL25 interaction by anti-CCL25 Ab markedly inhibited the development of experimental AIP (Figure 7, A and B). In addition, pancreatic expression of IFN-α, IL-33, TNF-α, and IL-6 was markedly decreased following administration of anti-CCL25 Ab as compared with administration of control Ab (Figure 7C). Finally, consistent with these changes in cytokine profiles, treatment with anti-CCL25 Ab significantly reduced the percentages of pDCs, CD11b+ myeloid cells, and CD11c+ DCs as compared with the administration of control Ab; in addition, such treatment was accompanied by CD11b+ myeloid cells and CD11c+ DCs that tended to have reduced CD11b and CD11c expression, respectively (Figure 7D and Supplemental Figure 2B). Thus, these data suggest that pDC migration is necessary for the development of experimental AIP and that such migration is mediated by interaction between CCL9 and CCL25, but not that between CCR2 and CCL2.

Pancreatic accumulation of pDCs requires the molecular interaction between CCR9 and CCL25. MRL/MpJ mice were administered poly(I:C) twice a week for a total of 16 times by intraperitoneal injection. Each poly(I:C) injection was preceded by intraperitoneal injection of anti-CCL25 Ab (100 μg, n = 5) or control Ab (100 μg, n = 3). After sacrifice at 3 hours following the final set of injections, pancreases were removed and analyzed as indicated. (A and B) Representative hematoxylin and eosin staining of the pancreatic tissues and pathological scores of induced autoimmune pancreatitis in the 2 groups. Original magnification, ×400. (C) Concentrations of IFN-α, IL-33, TNF-α, and IL-6 in protein extracts of pancreatic tissues obtained from mice in the 2 groups. (D) Flow cytometric analyses showing the percentages of pDCs, CD3+ T cells, CD11b+ myeloid cells, and CD11c+ DCs among pancreatic mononuclear cells. Left panels: Representative flow cytometric analyses. Right panels: Bar graphs of cumulative data from individual mice. pDCs were defined as PDCA-1+B220lo cells. Each dot represents the value derived from 1 mouse. Statistical analyses were performed using an unpaired, 2-tailed Student’s t test. Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01.

Pancreatic CD3+ T cells stimulated by IFN-α are the source of CCL25 production. Having obtained evidence that interaction between CCR9 and CCL25 plays an important role in the migration of pDCs into the pancreas, we conducted studies to identify the cellular source of CCL25 and the cytokines inducing its secretion. Here, we first performed an immunofluorescence analysis of inflamed pancreatic tissue from mice administered repeated 16 doses of poly(I:C) in the usual AIP induction regimen and found that cells bearing surface CD3 (i.e., CD3+ T cells) express intracellular CCL25 (Supplemental Figure 3C), whereas amylase-positive cells (pancreatic acinar cells) do not express CCL25 (A Hara, unpublished observation). We then determined the cell and cytokine environment supporting such CD3+ T cell CCL25 production. For this purpose, we examined pancreatic tissues from mice administered repeated 16 doses of poly(I:C) in the usual AIP induction regimen, but in this case in combination with anti-pDC Ab (120G8 Ab), anti–IFN-α/β receptor Ab (anti-IFNAR Ab), anti-CXCR3 Ab, and anti–IL-33 receptor Ab (anti-ST2 Ab) and then evaluated the expression of CCL25 in pancreatic tissues by blindly counting immunoreactive CCL25+ cells that were morphologically identified as lymphoid cells (4, 5). We found that the number of such cells positive for CCL25 was markedly decreased by treatment with 120G8 Ab, anti-IFNAR Ab, or anti-CXCR3 Ab, but not decreased by treatment with anti-ST2 Ab upon the blinded study (Figure 8, A–D). Thus, CD3+ T cells bearing CXCR3 and stimulated by IFN-α, but not IL-33, are the source of CCL25 production.

Pancreatic lymphoid cells stimulated by IFN-α are the source of CCL25. MRL/MpJ mice were administered poly(I:C) twice a week for a total of 16 times by intraperitoneal injection. Each poly(I:C) injection was preceded by intraperitoneal injection of (A) pDC-depleting Ab (120G8 Ab, 100 μg, n = 5) or control Ab (100 μg, n = 5); (B) anti–IFN-α/β receptor Ab (anti-IFNAR Ab, 100 μg, n = 5) or control Ab (100 μg, n = 4); (C) anti–IL-33 receptor Ab (anti-ST2 Ab, 100 μg, n = 5) or control Ab (100 μg, n = 5); and (D) anti-CXCR3 Ab (200 μg, n = 7) or control Ab (200 μg, n = 9). After sacrifice at 3 hours following the final set of injections, pancreases were removed and processed for immunohistochemical studies with anti-CCL25 Ab. Upper panels: Representative tissue images of CCL25 expression by morphologically identified lymphoid cells (indicated by arrowheads) in tissues obtained from mice administered control Ab or depleting/neutralizing Ab. Original magnification, ×800. Lower panels: Bar graphs showing numbers of CCL25+ lymphoid cells/high-powered field (HPF) (counted blindly). Each dot represents the value derived from 1 mouse. Statistical analyses were performed using an unpaired, 2-tailed Student’s t test. Results are expressed as mean ± SEM. **P < 0.01.

Pathogenic pDCs in the inflamed pancreas produce high levels of IFN-α when stimulated by CXCR3+ T cells producing CCL25. Whereas the initial source of IFN-α leading to influx of CCL25-producing T cells are TLR3+ cDCs responding to poly(I:C), once T cells are attracted into the inflamed pancreatic tissue and draw pDCs into this tissue, it is likely that the latter cells become the chief source of IFN-α. To examine this possibility, we first conducted in vitro studies utilizing flow cytometry–sorted CD3+CXCR3+ T cells, CD3+CXCR3– T cells, and pDCs obtained from inflamed pancreatic tissues of mice administered 16 doses of poly(I:C) to examine production of CCL25 and IFN-α within the mature inflamed pancreas (Figure 9A). We found that anti-CD3 Ab–stimulated CD3+CXCR3+ T cells secreted significantly higher levels of CCL25 than anti-CD3 Ab–stimulated CD3+CXCR3- T cells and in both cases, such secretion was greatly augmented by the presence of pDCs, particularly when the latter cells were stimulated by TLR9 ligand (CpG) (Figure 9B). In addition, pDC augmentation was substantially blocked by anti-IFNAR Ab, indicating that this effect was dependent on IFN-α production by pDCs (Figure 9, B and C). In line with these findings, CpG-stimulated pDCs secreted large amounts of IFN-α when cocultured with CD3+CXCR3+ T cells, but not CD3+CXCR3– T cells (Figure 9B). Thus, these in vitro studies show that CD3+CXCR3+ T cells are the source of high-level CCL25 secretion in the inflamed pancreas and that such secretion is dependent on pDC secretion of IFN-α.

CD3+CXCR3+ T cells produce CCL25 in response to IFN-α secreted by pDCs. (A) FACS gating strategy for acquisition of CD3+CXCR3+ T cells, CD3+CXCR3– T cells, and PDCA-1+B220lo pDCs. (B and C) Pancreatic mononuclear cells (PMNCs) were isolated from pancreases of MRL/MpJ mice (n = 3) administered poly(I:C) twice a week for a total of 16 times. PMNCs were subjected to FACS to acquire purified CD3+CXCR3+ T cells, CD3+CXCR3– T cells, and pDCs, after which they were cultured or cocultured for 48 hours (at 1 × 105/mL) in triplicate in the presence of anti-CD3 Ab (5 μg/mL) alone or with CpG (1 μM); in addition, in some experiments, anti–IFN-α/β receptor Ab (anti-IFNAR Ab, 100 μg/mL) or control Ab was added to the culture. Cell culture supernatants from each well were analyzed by ELISA for measurement of CCL25 and IFN-α. Each dot represents the CCL25 or IFN-α concentration value derived from each well. Statistical analyses were performed using the Kruskal-Wallis test and Bonferroni-corrected Mann-Whitney U test (B) or an unpaired, 2-tailed Student’s t test (C). Results shown are the representative 1 of 2 independent experiments and are expressed as mean ± SEM. **P < 0.01.

We next conducted in vitro studies utilizing flow cytometry–sorted CD3+CXCR3+ T cells, CD3+CXCR3– T cells, and cDCs obtained from pancreatic tissues of mice administered 3 poly(I:C) doses to examine CD3+ T cell production of CCL25 during the induction phase of experimental AIP (Supplemental Figure 4A). We found that CD3+CXCR3+ T cells produced greater amounts of CCL25 than CD3+CXCR3– T cells upon stimulation with anti-CD3 Ab in the presence of cDCs (Supplemental Figure 4B). In addition, the CCL25 production by CD3+CXCR3+ T cells was dependent on IFN-α since the addition of anti-IFNAR Ab reduced CCL25 production (Supplemental Figure 4B). It is worth noting that the amounts of CCL25 derived from CD3+CXCR3+ T cells in the presence of cDCs were smaller than those from the same T cell population in the presence of pDCs (Figure 9B and Supplemental Figure 4B).

In a final set of studies along these lines, we examined whether pDC production of IFN-α and IL-33 was influenced by interaction with activated T cells within the inflamed pancreas. To this end, pDCs from the pancreas of nontreated and poly(I:C)-treated MRL/MpJ mice and CD3+CXCR3+ T cells and CD3+CXCR3– T cells from the pancreas of poly(I:C)-treated MRL/MpJ mice were isolated by flow cytometry and then cocultured as indicated in Figure 9 in the presence or absence of anti-CD3 Ab or CpG. We found that pDCs isolated from the inflamed pancreas of mice secreted greater amounts of IFN-α and IL-33 than pDCs isolated from the uninflamed pancreas of mice when cultured in the presence of CpG (Figure 10A). In addition, pDCs isolated from inflamed pancreas, but not uninflamed pancreas, exhibited greatly increased IFN-α secretion and substantially increased IL-33 secretion when cocultured with activated CD3+CXCR3+ T cells rather than with CD3+CXCR3– T cells (Figure 10A). Finally, production of CCL25 was enhanced in a coculture composed of CD3+CXCR3+ T cells and pDCs isolated from the pancreas displaying established AIP (Figure 10A). These findings suggest that residency in the inflamed pancreas induces changes in the pDCs that allow enhanced costimulation by CD3+CXCR3+ T cells and greater responsiveness to TLR9 stimulation. Such enhanced costimulation may consist in an increased responsiveness to CCL25 that is being produced by cross-stimulated (IFN-α–stimulated) CD3+CXCR3+ T cells by the activated pDCs (Figure 9C and Figure 10A).

CD3+CXCR3+ T cells enhance differentiation of pDCs producing IFN-α and IL-33. (A) Purified CD3+CXCR3+ T cells, CD3+CXCR3– T cells, and pDCs were acquired from the pancreatic tissues of MRL/MpJ mice administered poly(I:C) (n = 3) or untreated MRL/MpJ mice (n = 3) and cocultured as described in Figure 9 in triplicate under the indicated conditions. Cell culture supernatants from each well were analyzed by ELISA for measurement of CCL25, IFN-α, and IL-33. Each dot represents the value derived from 1 well. Statistical analyses were performed using the Kruskal-Wallis test and Bonferroni-corrected Mann-Whitney U test. Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01. NS, not significant. (B) Diagram illustrating the positive feedback loop consisting of IFN-α/β, CXCL9, CXCL10, and CCL25 that establishes and sustains experimental pDC-driven autoimmune pancreatitis (see text for full description).

Taken together, these data suggest first that TLR9 ligand–stimulated pDCs in the inflamed pancreas are remarkably good producers of both IFN-α and IL-33 when present in a milieu containing CD3+CXCR3+ T cells. Second, they suggest that pDCs in the inflamed pancreas have a heightened capacity to respond to CpG following interaction with CD3+CXCR3+ T cells secreting CCL25. These findings, along with the other findings discussed above, allow us to define a cytokine/chemokine cascade or positive feedback loop that establishes the cellular elements necessary for pancreatic inflammation in the MRL/MpJ model of AIP (Figure 10B). This feedback loop is initiated by sensing of poly(I:C) by TLR3-bearing cDCs that induce differentiation of Th1 cells in draining lymphoid tissues and pancreatic expression of CXCL9 and CXCL10, chemokines that mediate migration of induced CXCR3+ Th1 cells into the pancreas. Pancreatic CXCR3+ T cells then produce CCL25 in response to further stimulation by IFN-α that, in turn, induces recruitment of CCR9+ pDCs into the pancreas. Finally, the pDCs now resident in the pancreas and costimulated by CXCR3+ T cells as well as TLR7/9 ligands become the enduring source of IFN-α that reinitiates a T cell–CCL25-pDC–IFN-α loop capable of sustaining AIP/IgG4-RD.

AIP/IgG4-RD in humans is marked by enhanced levels of serum CXCL9, CXCL10, and CCL25 that correlate with extent of disease. In the final series of the experiments, we investigated the question of whether the chemokines comprising the positive feedback loop noted above and responsible for pancreatic pDC and CXCR3+ T cell infiltration in the MRL/MpJ model of AIP are also present in human AIP/IgG4-RD. For this purpose, we determined and compared relevant chemokine concentrations in serum samples collected from healthy controls (n = 8) as well as patients with chronic alcoholic pancreatitis (CP) (n = 12), and AIP/IgG4-RD patients (n = 33) that met the established diagnostic criteria for these disorders (7). We found that serum concentrations of CXCL9, CXCL10, and CCL25 were markedly higher in patients with AIP/IgG4-RD as compared with those with CP and healthy controls, whereas in contrast, serum concentrations of CCL22, a prototypical Th2 chemokine, were not elevated in patients with AIP/IgG4-RD (Figure 11A). We next examined whether serum concentrations of CXCL9, CXCL10, and CCL25 are useful for the assessment of disease activity. Here, we measured serum concentrations of these chemokines before and after successful treatment with prednisolone (PSL). We found successful treatment in patients with AIP/IgG4-RD was associated with a significant reduction in serum concentrations of CXCL9, CXCL10, and CCL25 (Figure 11B). Thus, experimental and human AIP/IgG4-RD are both characterized by enhanced expression of CXCL9, CXCL10, and CCL25 and such enhanced expression is attenuated by treatment.

Serum concentrations of CXCL9, CXCL10, CCL25, and CCL22 in patients with autoimmune pancreatitis/IgG4-related disease. Serum samples were collected from healthy controls (HC, n = 8), chronic alcoholic pancreatitis (CP) patients (n = 12), and autoimmune pancreatitis (AIP)/IgG4-related disease (IgG4-RD) patients (n = 33). (A) Serum concentrations of CXCL9, CXCL10, CCL25, and CCL22; each dot corresponds to a value in 1 patient. Statistical analyses: Kruskal-Wallis test and Bonferroni-corrected Mann-Whitney U test. Results are expressed as mean ± SEM. (B) Serum chemokine levels from patients with AIP/IgG4-RD (n = 14) before and after induction of remission with prednisolone (PSL) therapy. Statistical analyses: Wilcoxon’s signed-rank test. (C and D) Correlation between serum IgG4 or IFN-α levels and chemokines in patients with AIP/IgG4-RD. Each dot represents 1 patient. P values and correlation coefficient (r) values, as determined by Spearman’s rank correlation test, are shown. *P < 0.05; **P < 0.01.

In previous studies, we reported that serum concentrations of IgG4, IFN-α, and IL-33 were useful biomarkers for diagnosis of AIP/IgG4-RD and monitoring of disease activity (7). We therefore compared levels of these cytokines with levels of relevant chemokines to determine whether the latter might also serve as biomarkers. We found that serum concentrations of IgG4 were positively correlated with those of CXCL9 and CXCL10, but not of CCL25 or CCL22 (Figure 11C). In addition, we found a positive correlation between the serum concentrations of IFN-α and CXCL9 or CXCL10, but no significant correlation between serum concentrations of IFN-α with those of CCL25 or CCL22 (Figure 11D). Finally, we found a positive correlation of serum concentrations of IgG1 and IL-33 with those of CXCL9 or CXCL10 (Supplemental Figures 5 and 6, respectively). Thus, these serum chemokine analyses suggest that serum concentrations of CXCL9, CXCL10, and CCL25 might be useful biomarkers for the diagnosis and monitoring of disease activity in AIP/IgG4-RD.