|PD-L2 modulates asthma severity by directly decreasing dendritic cell IL-12 production.|
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|PMID: 23149662 Owner: NLM Status: Publisher|
|Studies examining the role of programmed death 1 (PD-1) ligand 2 (PD-L2)/PD-1 in asthma have yielded conflicting results. To clarify its role, we examined the PD-L2 expression in biopsies from human asthmatics and the lungs of aeroallergen-treated mice. PD-L2 expression in bronchial biopsies correlated with the severity of asthma. In mice, allergen exposure increased PD-L2 expression on pulmonary myeloid dendritic cells (DCs), and PD-L2 blockade diminished allergen-induced airway hyperresponsiveness (AHR). By contrast, PD-1 blockade had no impact, suggesting that PD-L2 promotes AHR in a PD-1-independent manner. Decreased AHR was associated with enhanced serum interleukin (IL)-12 p40, and in vitro stimulation of DCs with allergen and PD-L2-Fc reduced IL-12 p70 production, suggesting that PD-L2 inhibits allergen-driven IL-12 production. In our model, IL-12 did not diminish T helper type 2 responses but rather directly antagonized IL-13-inducible gene expression, highlighting a novel role for IL-12 in regulation of IL-13 signaling. Thus, allergen-driven enhancement of PD-L2 signaling through a PD-1-independent mechanism limits IL-12 secretion, exacerbating AHR.Mucosal Immunology advance online publication 14 November 2012. doi:10.1038/mi.2012.111.|
|I P Lewkowich; S Lajoie; S L Stoffers; Y Suzuki; P K Richgels; K Dienger; A A Sproles; H Yagita; Q Hamid; M Wills-Karp|
|Type: JOURNAL ARTICLE Date: 2012-11-14|
|Title: Mucosal immunology Volume: - ISSN: 1935-3456 ISO Abbreviation: Mucosal Immunol Publication Date: 2012 Nov|
|Created Date: 2012-11-14 Completed Date: - Revised Date: -|
Medline Journal Info:
|Nlm Unique ID: 101299742 Medline TA: Mucosal Immunol Country: -|
|Languages: ENG Pagination: - Citation Subset: -|
|Division of Cellular and Molecular Immunology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.|
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Journal ID (nlm-journal-id): 101299742
Journal ID (pubmed-jr-id): 35518
Journal ID (nlm-ta): Mucosal Immunol
Journal ID (iso-abbrev): Mucosal Immunol
nihms-submitted publication date: Day: 15 Month: 12 Year: 2012
Electronic publication date: Day: 14 Month: 11 Year: 2012
Print publication date: Month: 7 Year: 2013
pmc-release publication date: Day: 01 Month: 1 Year: 2014
Volume: 6 Issue: 4
First Page: 728 Last Page: 739
PubMed Id: 23149662
|PD-L2 modulates asthma severity by directly decreasing dendritic cell IL-12 production|
1Division of Cellular and Molecular Immunology, Cincinnati Children’s Hospital Medical Center, Department of Pediatrics, Cincinnati, OH
2Bloomberg School of Public Health, John Hopkins University, Baltimore, MD
3Division of Pulmonary Medicine, Department of Medicine, Keio University School of Medicine, Tokyo, Japan
4Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan
5Meakins-Christie Laboratories, Faculty of Medicine, McGill University, Montreal, Quebec, Canada
|Correspondence: Address correspondence to: Ian Lewkowich, Ph.D., Division of Cellular and Molecular Immunology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, Ph: (513)-636-3999
Allergic asthma is an inflammatory lung disease whose prevalence continues to rise in developed nations. Although the origins of allergic asthma are complex, excessive activation of Th2 cells specific for normally innocuous environmental allergens drives disease pathology. Thus, in asthmatic individuals, allergen exposure triggers the development of allergen-specific Th2 cells producing IL-4, IL-5 and IL-13, which induce IgE, eosinophilia, mucus hypersecretion, and airway hyperresponsiveness (AHR)1. In contrast, non-asthmatic individuals develop protective responses mediated by IL-10 and TGF-β secreting Tregs2-3. Thus, the type of T cell response induced following allergen exposure is an important determinant of susceptibility to allergen-induced AHR.
In vivo, dendritic cells (DCs) represent the most potent professional antigen presenting cells in the lung, and they have been identified as key players in promoting both pathogenic or protective T cell responses4. In the lung, DCs lie immediately below the epithelial cell layer and extend their dendrites into the lumen, capture inhaled allergens, and then migrate to lung-draining lymph nodes (LNs) where they present captured allergens to naive T cells5-6. Many factors influence the ability of DCs to promote pathogenic versus protective T cell responses. Allergen presentation by myeloid DCs (mDCs) promotes both initiation of AHR in naive animals4, 7 and exacerbations in previously sensitized animals6, while allergen presentation by plasmacytoid DCs (pDCs) prevents the initiation of AHR8 and limits allergic inflammation in previously sensitized hosts9. DCs also influence the development of allergen-induced AHR through production of T cell skewing cytokines. We have recently demonstrated that the production of high levels of IL-23 by pulmonary mDCs is associated with the development of a mixed Th2/Th17 cytokine profile that ultimately results in the development of severe AHR4, 10. In contrast, the development of weak AHR observed in C3H/HeJ mice is associated with the production of IL-1211, and administration of exogenous IL-12 limits the severity of AHR in asthma-susceptible A/J animals12. Finally, the types of co-stimulatory molecules expressed by DCs also control T cell development such that expression of CD80, CD86 and OX40L all promote allergic disease13-15, while ICOS-L expression induces the development of Tregs and promotes inhalational tolerance16. Thus, by virtue of the subsets of DCs involved in allergen presentation and the nature of the cytokines or co-stimulatory molecules produced, DCs regulate the development of pathogenic versus protective T cell responses.
Another way DCs regulate T cell responses is through expression of PD-1 family members. The PD-1 family consists of PD-L1 (found on DCs, T cells, B cells, and structural cells17-18), PD-L2 (expressed on DCs and macrophages18) and PD-1 (expressed on T cells, DCs and macrophages19-21). In general, stimulation of T cells through PD-1 or PD-L1 limits CD3-mediated activation of T cells. Indeed, PD-1 stimulation is responsible for the T cell “exhaustion” observed in models of chronic viral infection (LCMV, HIV)22-23, contributes to allograft tolerance24, and prevents autoimmune disease17, 25. Interestingly, despite the generally inhibitory role for PD-1 in regulating T cell activation, the role of the PD-1/PD-L axis in regulating allergic asthma is uncertain, with some reports suggesting a protective role for this pathway9, 26-29, and others reporting a pro-asthmatic role for either PD-L127 or PD-L230.
Herein, we sought to clarify the role of PD-L2 in allergen-induced AHR. We demonstrate that in both mice and humans, increased PD-L2 expression is associated with more severe AHR. Supporting a role for a PD-L2 in modulating asthma severity, in vivo blockade of PD-L2 reduces the severity of allergen-driven AHR. Surprisingly, PD-L2 blockade does not alter the production of Th2 cytokines, the composition of inflammatory cells in the bronchoalveolar lavage, or synthesis of IgE, but rather, enhances the ability of DCs to secrete IL-12 p70, and simultaneous blockade of both PD-L2 and IL-12 abrogate the protective effects observed upon blockade of PD-L2 alone. Strikingly, blockade of PD-1 had no impact on AHR, or systemic IL-12 levels, suggesting that PD-L2 regulates asthma severity in a PD-1 independent manner. Furthermore, in our model, increased IL-12 production is not associated with decreased Th2 cytokine production. However, we demonstrate that IL-12 directly antagonizes IL-13-driven gene induction and STAT6 phosphorylation, providing a mechanism whereby IL-12 may limit the severity of allergen-induced AHR. Collectively, these results demonstrate that PD-L2, through an unknown receptor, limits IL-12 secretion, thereby enhancing the magnitude of allergen-induced AHR.
In this study, we sought to clarify the role of PD-L2 in asthma. To this end, we compared PD-L2 expression in the lung following sequences of allergen exposures designed to induce either robust AHR, or the development of tolerance. As such, we exposed mice to house dust mite extract (HDM) i.t. on days 0 and 14, a protocol previously shown to induce robust AHR in A/J mice4. As expected, HDM exposure induced robust AHR (Fig 1a) as measured by monitoring airway pressure changes over time (APTI), as described elsewhere31. We then examined PD-L2 mRNA expression in the lungs of mice at 2, 24 or 72 hours after the last i.t. challenge. PD-L2 expression was up-regulated by HDM at 2 hours and peaked 24 hours later (Fig 1b). To determine if PD-L2 induction was unique to HDM, A/J mice were sensitized i.p. with OVA in saline on day 0, and challenged i.t. with OVA on days 14 and 21. As previously reported11, this treatment regimen induced AHR (Fig 1a). As with HDM, OVA exposure also induced a rapid increase in PD-L2 expression that peaked at 24 hours (Fig 1b). Interestingly, despite an identical number of allergen exposures, mice receiving OVA exclusively i.t., a regimen known to induce tolerance rather than AHR8, 16, demonstrated no induction of AHR (Fig 1a) and failed to up-regulate PD-L2 expression significantly, suggesting that enhanced PD-L2 expression is associated with allergen-induced AHR (Fig 1b).
PD-L2 is chiefly expressed by antigen presenting cells18, however, multiple pulmonary DC populations have been described to date. To determine if PD-L2 was differentially expressed on different DC subsets, mice were treated i.t. with HDM on day 0, and fluorescent HDM (AF405-HDM) on day 14 to facilitate the identification of allergen-bearing DCs by flow cytometry. 72 hours after HDM exposure 5 distinct populations of CD11c+ cells could be identified by flow cytometry, including alveolar macrophages (AMs), plasmacytoid DCs (pDCs), neutrophils, inflammatory DCs, and myeloid DCs (mDCs) (Supplementary Fig 1a). While AMs demonstrated some allergen uptake, and AMs, neutrophils and inflammatory DCs all expressed some PD-L2 after allergen challenge, mDCs were the only population simultaneously positive for both AF405-HDM and PD-L2 (Supplementary Fig 1b). Thus, HDM+PD-L2+ mDCs represented up to ~15% of all mDCs found in the lung, while the increase in the frequency of other HDM+PD-L2+ DC populations was comparatively minor (≤ 3%) (Fig 1c). Moreover, the levels of PD-L2 expression (as measured by MFI) increased markedly on pulmonary mDCs after allergen exposures (Fig 1d). In contrast the MFI of PD-L2 expression was not significantly increased on other pulmonary DC populations (Fig 1d). These data suggest that pulmonary mDCs are the principal PD-L2 expressing DC subset in the lung.
To determine if a similar association between PD-L2 expression and increased asthma severity is present in human populations, we compared PD-L2 expression in bronchial biopsies from patients with mild asthma, severe asthma, and healthy control subjects. PD-L2 expression was undetectable in healthy control subjects (Fig 1e). In comparison to healthy controls, the expression of PD-L2 was enhanced in patients with severe asthma (p < 0.05) (Fig 1e). Moreover, while not significant, there was a trend towards increased PD-L2 expression in individuals with mild asthma (p = 0.07). Thus, PD-L2 expression is significantly elevated in individuals with severe disease.
To directly assess the role of PD-L2 in AHR in mice, we examined the effect of a PD-L2 blocking mAb (clone TY25)32 on the development of allergen-induced AHR. As expected, HDM increased AHR, which was not impacted by an isotype control mAb (Fig 2a). In contrast, administration of anti-PD-L2 significantly reduced the development of HDM-induced AHR, demonstrating that PD-L2 modulates asthma severity (Fig 2a). While PD-L2 is a ligand for PD-133, some reports suggest that PD-L2 can bind a receptor other than PD-134-35. To determine if PD-L2 enhances asthma through interactions with PD-1, we examined the impact of PD-1 blockade on the development of AHR. However, treatment with a mAb known to block interactions between PD-1 and both of its known ligands (PD-L1 and PD-L2) (clone RMP1-14)36 had no impact on the magnitude of HDM-induced AHR in A/J animals (Fig 2b). This suggests that PD-L2 regulates the severity of AHR through a PD-1 independent mechanism.
As airway inflammation is a hallmark of allergen-induced asthma, we also examined the cells present in the bronchoalveolar lavage (BAL) of mice following exposure to PBS, HDM, HDM + isotype control mAb, or HDM + anti-PD-L2. We harvested BAL at the time of sacrifice and performed differential cell counts. As expected, HDM exposure induced the recruitment of significant numbers of macrophages, eosinophils, neutrophils and lymphocytes (Fig 3a). However, the BAL cell profile was unaffected by either the presence of the control mAb, anti-PD-L2 (Fig 3a) or anti-PD-1 (data not shown). Moreover, analysis of BAL cytokine levels demonstrated that while HDM increased the amounts of IL-4, IL-5 and IL-13 found in the BAL fluid, the levels of these cytokines were not significantly impacted by administration of isotype control, or PD-L2 blocking mAbs (Fig 3b). To further characterize the immune response, we also examined HDM-stimulated cytokine production in lung cell cultures from mice treated in vivo with anti-PD-L2 or a control mAb. As previously shown, HDM-treated A/J mice display a mixed Th2/Th17 cytokine profile, with robust production of IL-4, IL-5, IL-13, and IL-17A (Fig 3c-f), but no IFNγ (data not shown). However, we observed no impact of in vivo anti-PD-L2 treatment on the production of any of the cytokines examined. Consistent with unaltered T cell cytokine production or BAL cell recruitment, the activation status of pulmonary myeloid DCs was also unaltered in mice treated with anti-PD-L2 (Supplementary Fig 2). Thus, in our model, endogenous expression of PD-L2 appears to regulate the severity of allergen-induced AHR, but not by amplifying the intensity of the underlying Th2 or Th17 responses.
As mucus hypersecretion is also a hallmark of allergic asthma, we assessed mucus production in the lungs of mice treated with PD-L2 blocking Ab. As expected, compared to mice treated with PBS, those treated with HDM + Isotype demonstrated markedly higher numbers of epithelial cells that stained with PAS (Fig 4a). In contrast, treatment of HDM-exposed mice with anti-PD-L2 significantly decreased the number of PAS+ epithelial cells (Fig 4a). To further characterize the immune response in HDM + anti-PD-L2-treated A/J mice, we assessed serum immunoglobulin levels. HDM exposure induced increased total IgE (Fig 4b) (as a surrogate for allergen-specific IgE activity37), HDM-specific IgG1 (Fig 4c), and HDM specific-IgG2a (Fig 4d), but treatment with control mAb had no impact on serum Ig levels. While PD-L2 blockade did not alter IgE or IgG1 production, PD-L2 blockade nearly doubled the levels of HDM-specific IgG2a. As IgG2a production is driven by Th1-associated cytokines, we examined levels of IL-12 p40 in the serum (Fig 4e). IL-12 p40 levels in the serum were significantly enhanced in HDM + anti-PD-L2 treated mice, suggesting that PD-L2 can limit IL-12 p40 levels (Fig 4f). Interestingly, a similar increase was not observed in BAL, where IL-12 p40 levels were unaffected by anti-PD-L2 treatment (Supplementary Fig 3). As increases in systemic IL-12 levels limit the development of allergen-induced AHR in A/J mice12, the increase in serum IL-12 p40 may explain the decreased AHR observed in anti-PD-L2 treated mice.
To specifically test the hypothesis that increased IL-12 levels observed following PD-L2 blockade are responsible for decreased AHR, we treated mice with HDM in the presence of Isotype control mAbs, HDM + anti-PD-L2, HDM + anti-IL-12, or HDM + anti-PD-L2 + anti-IL-12. While treatment of A/J mice with anti-IL-12 had no impact on AHR, treatment of mice with anti-PD-L2 significantly diminished AHR (Fig 5a). However, treatment with anti-IL-12 abrogated the protective effect of PD-L2 blockade, as AHR in mice treated simultaneously with anti-PD-L2 and anti-IL-12 was indistinguishable from mice treated with isotype control mAbs alone (Fig 5a). Baseline AHR measurements (taken prior to injection of acetylcholine) demonstrated that while HDM increased basal AHR, this was not significantly different among treatment groups (Supplementary Fig 4).
Examination of HDM-restimulated cytokine production in lung cell cultures from anti-PD-L2, anti-IL-12 or anti-PD-L2 + anti-IL-12 treated mice revealed that while HDM induced production of both Th2 (IL-4, IL-5 and IL-13) (Fig 5 b-d) and Th17 (IL-17A) (Fig 5e) cytokines, there was no difference between treatment groups.
As IL-12 p40 is a component of both bioactive IL-12 p70 and IL-23, which may have opposing effects on the development of allergen-induced AHR4, 11, we next determined whether changes in IL-12 p40 observed following PD-L2 blockade were due to specific effects on IL-12 p70 or IL-23. To this end we cultured bone marrow-derived mDCs (BMDCs) with HDM on plates coated with PD-L2-Fc, PD-L1-Fc or a control IgG-Fc. HDM induced IL-12 p70 production from BMDCs, and this production was unaltered by IgG-Fc (Fig 6a). DCs cultured on PD-L2-Fc-coated plates produced significantly less IL-12 p70 (Fig 6a) suggesting that stimulation with PD-L2-Fc can limit HDM-induced IL-12 p70 production. In contrast, stimulation of DCs with HDM in the presence of PD-L1-Fc, an alternative ligand for PD-1, did not impair IL-12 p70 production, suggesting PD-1 signaling is not regulating IL-12 production. The inhibition was specific for IL-12, as stimulation with PD-L2-Fc did not alter HDM-induced secretion of other DC-derived cytokines IL-23, IL-6, TNFα (Fig 6b-d) or IL-10 (data not shown). To directly test whether the capacity of PD-L2 signaling to limit bioactive IL-12 production was independent of PD-1, we cultured BMDCs on plates coated with either rIgG-Fc, or PD-L2-Fc and stimulated these cells in the presence of anti-PD-1. As shown in Figure 6e, HDM-stimulated BMDCs plated on PD-L2 coated plates produced less IL-12 p70, and their overall production of IL-12 p70 was not affected by the presence of PD-1 blocking mAb. Similar results were observed when cells were stimulated with LPS (Fig 6f). Thus stimulation with PD-L2 appears to specifically limit the capacity of DCs to secrete IL-12 p70 through a PD-1 independent mechanism.
Our observation that enhanced IL-12 production was associated with diminished AHR and mucus secretion, but unaltered production of IL-4, IL-5, IL-13 and IL-17A suggests that IL-12 may be regulating the severity of AHR by directly antagonizing signaling initiated by Th2 cytokines. As IL-13 plays a major role in both allergen-induced AHR and mucus secretion38, we tested the hypothesis that IL-12 was antagonizing IL-13 signaling. To this end, BMDCs were cultured with medium, IL-12, IL-13 or IL-12 + IL-13 and phosphorylation of STAT6, a signaling intermediate directly activated as a result of IL-13R engagement, was measured by western blot. As shown in Figure 7a, no detectable STAT6 phosphorylation was observed in medium, or IL-12 stimulated BMDC cultures, while IL-13 induced robust STAT6 phosphorylation. In the presence of IL-12, IL-13 induced STAT6 phosphorylation was reduced, but not completely abrogated (Fig 7a). To confirm that the reduced STAT6 phosphorylation observed in IL-12 + IL-13 stimulated BMDC cultures was biologically significant, we subsequently examined the impact of IL-12 exposure on the expression of IL-13-driven genes by RT-PCR. While IL-13 induced the expression of FIZZ1, Arg1, CD206 in BMDCs (Fig 7b) the induction of these genes was reduced following co-culture with IL-12 (Fig 7b). As reported elsewhere28, IL-13 also induced the expression of PD-L2 on BMDCs. Interestingly, IL-13-driven PD-L2 expression was similarly inhibited in the presence of IL-12, suggesting that IL-12 may support its own production by antagonizing IL-13-driven PD-L2 expression. Not all IL-13 driven genes were inhibited to the same extent, as IL-12 completely abrogated CD206 expression, while effects on other IL-13 driven genes (Arg1, Fizz1, PD-L2) were only partially abrogated, consistent with reduced, but not abrogated, phosphorylation of STAT6 observed in Fig 6a. In contrast, while IL-12 increased IFNγ expression in A/J BMDCs, this was not antagonized by IL-13, suggesting that IL-13 may not antagonize IL-12 signaling (Supplementary Fig 6a). Moreover, other Th1 cytokines can have a similar impact, as co-culture of BMDCs with IFNγ and IL-13 also abrogated IL-13-driven gene expression (Supplementary Fig 6b). In contrast to the inability of IL-13 to inhibit IL-12-driven genes, IL-13 strongly inhibited the induction of an IFNγ-driven gene (PD-L1) (Supplementary Fig 6b).
As IL-12 is widely known as an inducer of IFNγ, we also determined if the inhibition of IL-13 signaling by IL-12 was mediated by IFNγ, through the blockade of IFNγ in cultures of BMDCs treated with IL-12 and IL-13. Inhibition of IL-13-induced Fizz1 expression by IL-12 was completely reversed by addition of anti-IFNγ (Supplementary Fig 6c). However it is unlikely that the effects of IL-12 on IL-13 signaling were entirely dependent upon induction of IFNγ, a s I L-12-mediated inhibition of STAT6 phosphorylation was observed as little as 15 minutes after IL-13 stimulation (too rapid to be due to de novo IFNγ synthesis) (Fig 7a), and IFNγ blockade did not restore expression of Arg1 or CD206 to levels seen in the absence of IL-12 (Supplementary Fig 6c).
As epithelial cell responsiveness to IL-13 is sufficient to observe IL-13 induced alterations in AHR, mucous cell hyperplasia and cell recruitment39, it is also important to assess the ability of IL-12 to inhibit IL-13 signaling in airway structural cells. To this end, primary, murine tracheal epithelial cells were stimulated with IL-12, IL-13, or IL-12 + IL-13 and expression of IL-13-driven genes was assessed by RT-PCR. As shown in Figure 7c, IL-13 induced upregulation IL-33, FIZZ1, AMCase, and SPRR2b. Consistent with the effects observed on BMDCs, IL-12 abrogated IL-13-driven induction of these genes in epithelial cells as well. Thus, IL-12 appears to directly inhibit IL-13-induced gene expression in both hematopoietic and structural cells through both IFNγ-dependent, and independent mechanisms. Collectively these data demonstrate that IL-12 may limit AHR by directly antagonizing signaling of Th2-associated cytokines.
In this study, we have examined the role of the co-stimulatory molecule, PD-L2, in the development of allergen-induced AHR. We find that PD-L2 is upregulated in the lungs of individuals with severe (but not mild) asthma and in allergen-exposed mice. In contrast to current dogma, which would suggest an inhibitory role for PD-L2 based on interactions with PD-1, in vivo PD-L2 blockade reduces allergen-induced AHR. While PD-L2 blockade reduced AHR, no significant impact on eosinophilia, IgE synthesis, or pulmonary dendritic cell activation was observed, suggesting that PD-L2 regulates AHR without impacting other features of allergic asthma. Collectively these data suggest that PD-L2 expression regulates the overall magnitude of AHR in allergic asthma.
Our observation that PD-L2 signaling exacerbates asthma severity is in agreement with the previous report that PD-L2-Fc treatment enhances pulmonary inflammation in OVA (alum) sensitized BALB/c mice30. However, in contrast to our results, it has also been reported that blockade of PD-L2 (but not PD-1) in OVA (alum) sensitized BALB/c mice triggers increased AHR, a phenotype that was blocked with co-administration of IFNγ-blocking antibodies26. Similarly, AHR induced by exposure of BALB/c mice to OVA (alum) or αGalCer was increased in PD-L2 deficient mice due to increased IL-4 production by iNKT cells27. While these reports appear to conflict with our own, both are consistent with an increase in IL-12 production in the absence of PD-L2 signaling. In the study by Matsumoto et al, while IL-12 levels were not assessed, it is likely that the IFNγ driving increased AHR following PD-L2 blockade was driven by enhanced IL-12 production triggered by the lack of PD-L2 signaling. Moreover, IL-12 has been shown to drive IL-4 production in iNKT cells40-41. Thus as PD-L2 −/− mice lack a negative regulator of IL-12 production, iNKT cells from these mice display increased production of IL-427. Thus, collectively these studies support an important role for PD-L2 in the direct regulation of IL-12 synthesis.
While IL-12 is classically regarded as a shaper of adaptive immunity, we find that the enhancement of IL-12 production observed following PD-L2 blockade does not regulate the balance of Th1 and Th2 associated cytokines in our model. It should be noted that while we observe robust PD-L2 expression after allergen challenge, we observe very few PD-L2+ mDCs immediately after a single allergen inhalation (data not shown). This suggests that the elevated IL-12 observed following anti-PD-L2 treatment is likely only present during the effector phase, after allergen challenge. Thus, by inhibiting IL-12 levels exclusively during the effector phase, we find that PD-L2 directly promotes IL-13-driven sequelae (AHR, mucus hypersecretion) in allergic asthma independently of its effects on T cell differentiation. While it is conceivable that blockade of PD-L2 induces other changes which may contribute to the reduced AHR observed, simultaneous blockade of PD-L2 and IL-12 abrogates the protective effects of PD-L2 blockade, suggesting that the increased IL-12 observed is the dominant influence on the reduced AHR observed following PD-L2 blockade.
While the precise gene products induced by IL-13 that promote the development of AHR are not clear, we have demonstrated that IL-12 can inhibit a number of IL-13-induced products with described roles in allergic asthma. In bone marrow-derived dendritic cells IL-12 inhibits IL-13-induced expression of Fizz1 and Arg1, products associated with alternatively activated macrophages which have been shown to play a pathogenic role in allergic asthma42-43. Additionally, IL-12 also antagonized the IL-13-induced expression of CD206 (a receptor shown to promote the phagocytosis of Derp144) in BMDCs. While we do not observe an increase in overall HDM uptake by pulmonary DCs following blockade of PD-L2 in vivo, it is possible that PD-L2 blockade may subtly regulate the ability of DCs to take up or present specific allergenic peptides. In primary murine tracheal epithelial cells, IL-12 also inhibited the IL-13-induced expression of the genes encoding IL-33, (a Th2-inducing cytokine45-46), AMCase (capable of enhancing allergen-driven IL-13 production47-48), and Fizz1 (capable of inducing airway eosinophilia and airway remodeling49). We also see reduced IL-13-driven expression of Sprr2b following IL-12 treatment. While little is known about the role of Sprr2b in the development of allergic asthma, it is induced by IL-13, but not IL-450. This is of particular interest as IL-13 is considerably more important than IL-4 in the induction of AHR38, suggesting that gene products induced by IL-13, but not IL-4 may have an important role in promoting AHR. Finally, we also demonstrate that IL-12 can directly antagonize STAT6 phosphorylation triggered by IL-13. It is likely that this effect may directly contribute to the reduced AHR observed in mice treated with anti-PD-L2 as STAT6 activation is critically required to trigger IL-13-induced AHR39, 51-52. Thus, despite unaltered levels of overall IL-13 production, IL-12 may impact the overall bioactivity of IL-13 through a number of different mechanisms.
That IL-12 may directly antagonize IL-13 signaling is also consistent with our previous observation that IL-12 blockade in A/J mice enhanced basal acetylcholine-induced airway constriction, suggesting that IL-12 can regulate airway tone in the absence of airway inflammation11 and with reports showing IL-12-deficient mice can display enhanced AHR, with minimal impact on Th2 cytokine production11, 53-54. We also find elevated PD-L2 expression in individuals with severe asthma, but not those suffering from milder forms of disease, suggesting that PD-L2 expression may also be correlated with asthma severity in humans as well. Although we did not examine IL-12 levels in individuals with severe versus mild asthma, Morahan et al. have shown that a combination of SNPs in the human IL-12 p40 promoter resulting in reduced LPS-stimulated IL-12 p40 secretion by PBMCs is associated with more severe asthma, regardless of the atopic status of the individual55. This suggests that impaired IL-12 production in humans also correlates with asthma severity, but not T cell skewing leading to initial sensitization (i.e. atopic status). Thus, our study highlights a previously underappreciated role of IL-12 in directly limiting asthma severity.
PD-L2 is typically thought to signal through PD-1 providing an inhibitory signal, limiting proliferation, and cytokine production of both T cells30, 56-58 and APCs20-21, 59. However interactions between PD-L2 and a receptor other than PD-1 were observed in a number of in vitro studies where it was demonstrated that such interactions enhance T cell activity33-35, 60. Multiple lines of evidence suggest that, in our model, the dominant signals provided by PD-L2 are mediated by a non-PD-1 receptor including: 1) in vivo blockade of PD-L2 and PD-1 have differential impacts on HDM-induced AHR, 2) stimulation with PD-L2, but not PD-L1, limits HDM- and LPS-induced IL-12 production in vitro, and 3) the ability of PD-L2 to inhibit HDM or LPS-driven IL-12 p70 production in BMDCs is not abrogated in the presence of antibodies known to block the interactions between PD-L2 and PD-1. As stimulation of DCs with PD-L2, but not PD-L1, limits HDM-induced IL-12 production, we speculate that DCs also express the unidentified PD-L2 receptor, and that signaling through this receptor in DCs directly regulates IL-12 p70 production. Intriguingly, as PD-L2 is primarily expressed by mDCs, the ability of PD-L2 to downregulate DC-derived IL-12 production suggests that autocrine or paracrine interactions between DCs are capable of regulating IL-12 production. Thus, PD-L2-mediated regulation of immune responses is complex, being regulated via interactions between PD-L2 and at least two distinct receptors, both of which are expressed on DCs and T cells, and which have distinct effects based on the types of cells studied. Such complexities suggest that PD-L2 (and other family members) acts as a rheostat, modulating the responsiveness to immune mediators such as IL-13, rather than providing an “on/off” switch for one particular type of response. However, our observation of differential expression levels of PD-L2 in human tissues taken from individuals with mild versus severe asthma, suggest that these subtle influences can have a substantial impact on disease severity.
Collectively these data demonstrate that PD-L2 expression modulates the overall magnitude of AHR in allergic asthma. However, rather than directly regulating T cell activity through interactions with its previously described receptor, PD-1, the dominant function of PD-L2 in vivo is inhibition of APC-derived IL-12 through interactions with an unknown receptor. Moreover, PD-L2 stimulation of purified DCs directly inhibits IL-12 production, demonstrating that the unknown receptor is also expressed by DCs and suggesting that DC:DC interactions, either autocrine or paracrine, modulate IL-12 synthesis in vivo. Finally, these data also demonstrate that IL-12 can directly antagonize IL-13-driven gene transcription, highlighting a previously unappreciated role for IL-12 in modulating the activity of Th2 effector cytokines. Thus, in asthmatics, increased PD-L2 expression in the inflamed lung downregulates IL-12 secretion, facilitating greater IL-13 responsiveness and allowing the development of more severe airway hyperresponsiveness.
Male A/J mice were purchased from Jackson Laboratories (Bar Harbor, ME) at 4 weeks of age and housed in a specific pathogen-free facility at Cincinnati Children’s Hospital Medical Center. Experiments were initiated at 6 weeks of age. Animal experiments were approved by the CCHMC Institutional Animal Care and Use Committee.
Mice were treated with 40 μl PBS, HDM (100 μg; Greer Laboratories, Lenoir, NC), or AlexaFluor405-labeled (Invitrogen, Carlsbad, CA) HDM (AF405-HDM) i.t. on days 0 and 14. Where indicated mice were treated with low-endotoxin OVA (Worthington Biochemicals, Lakewood, NJ) to induce AHR (10 μg i.p. on day 0, and 100 μg i.t. on days 14 and 21) or tolerance (100 μg i.t. on days 0, 14 and 21). Mice were treated intraperitoneally with 250 μg anti-PD-L2 (TY2518, 26), anti-PD-1 (RMP1-1436) or control rat IgG2a (GL117) 6 hours before, and 48 hours following each allergen exposure. Where indicated mice were also treated with 1 mg of anti-IL-12 (clone C17.8, a gift of Dr. Fred Finkelman, University of Cincinnati) or control rat IgG2a (GL117) on days −2 and 12.
Airway hyperresponsiveness (AHR) was measured by assessing changes in tracheal pressure following administration of a cholinergic agonist over time and expressed as the airway pressure time index (APTI), and is described in detail elsewhere31. Importantly, this parameter has been shown to provide data similar to Flexivent-derived data generated using a full dose response of inhaled methacholine61 To evaluate AHR, mice were anaesthetized, intubated and respirated at a rate of 120 breaths per minute with a constant tidal volume (0.2 ml) and paralyzed with decamethonium bromide (25 mg/kg) 72 hours after final allergen challenge. Acetylcholine (50 μg/kg) was injected into the inferior vena cava and dynamic airway pressure (cmH20xSec) followed for 5 minutes. Blood was collected to measure total IgE and HDM-specific IgG1 and IgG2a. To collect BALF, lungs were lavaged with HBSS (Invitrogen, Carlsbad, CA). Cells were recovered by centrifugation and total cells counted. Slides were prepared by cytocentrifugation and stained with Diff-Quik (Dade Behring, Düdingen, Switzerland) for differential cell counts.
After AHR measurements, lungs were removed, minced and placed in 6 ml of RPMI 1640 containing Liberase CI (0.5 mg/ml)(Roche Diagnostics) and DNase I (0.5 mg/ml)(Sigma) at 37°C for 45 minutes. The remaining tissue was forced through a 70-micron cell strainer, and red blood cells were lysed with ACK lysis buffer (Invitrogen). Cells were washed with RPMI containing 10% FBS, viable cells were counted via trypan blue exclusion. Where indicated, lung cells were cultured at 350,000 cells per well of a 96-well plate (250 μl final volume) with HDM (30 μg/ml). Tissue culture supernatants were harvested at 72 hours.
Staining reactions were performed at 4°C following incubation with FcBlock (mAb 2.4G2) for 30 minutes. Alveolar Macrophages (AM - CD11cbright, CD11bneg, CD317neg), plasmacytoid DCs (pDCs - CD11cdimCD11bnegCD317+), neutrophils (PMNs - CD11cbrightCD11bbrightGr1brightCD103neg), inflammatory DCs (Inf DCs - CD11cbrightCD11bbrightGr1dimCD103neg) and myeloid DCs (CD11cbrightCD11bbrightGr1negCD103neg) were quantified using anti-CD11c-PerCPCy5.5 (HL3), anti-CD11b-PE-Cy7 (M1/70), anti-Gr1-APC-Cy7 (RB6-8C5), anti-CD103-FITC (2E7), and anti-CD317-AF647 (eBio129c). Co-stimulatory molecule expression was examined using PE-conjugated mAbs to MHC Class II (14-4-4s) and PD-L2 (TY25). mAbs were purchased from eBioscience. Antigen uptake was measured in the Pacific Blue channel following administrated of AF405-conjugated HDM. Dead cells were excluded using Live/Dead Blue (Invitrogen). Data were acquired with an LSRII flow cytometer equipped with lasers tuned to 355 nm, 405nm, 488nm, 562nm, and 640nm, and digital DiVa Software. FlowJo software (Treestar Inc., Ashland, OR) used to compensate spectral overlap and analyze data.
Subjects with no asthma, or well-defined mild and severe asthma were recruited from the Montreal Chest Institute and Sacré-Coeur Hospital (Montreal, Quebec, Canada) based on the criteria of the ATS workshop for Refractory Asthma62. As described elsewhere63, mild asthmatics had FEV1 readings greater than or equal to 70% postbronchodilator and required infrequent use of inhaled short-acting β2-agonist medication alone. Severe asthmatics had at least one of (1) treatment with daily oral steroids for more than 50% of the previous 12 months, or (2) treatment with high-dose inhaled steroids and at least one other add-on therapy continuously over the past 12 months. In addition, 2 or more of the following were required: (1) daily use of short-acting β-agonist; (2) persistent airflow obstruction as documented by prebronchodilator FEV1 less than 70% and FEV1/FVC ratio less than 80% predicted; (3) at least 1 urgent care visit in the previous 12 months; (4) ≥3 steroid bursts in the previous 12 months; (5) prompt deterioration with less than 25% dose reduction of steroids; or (6) a near-fatal asthma event in the last 3 years. Detailed definition of the inclusion and exclusion criteria for the enrollment of asthmatic subjects were reported previously63. The protocols for the study were reviewed and approved by the ethics committees of both hospitals, and informed consent was obtained from all participating subjects. RNA was extracted from lung biopsies using the RNeasy columns, reverse transcribed with oligo(dT) (Amersham) and Superscript II (Invitrogen), in the presence of RNAguard (Amersham).
PCR primer pairs for mPD-L2, mFIZZ1, mArg1, mSPRR2b, mCD206, mC3, mS14, mIL-5, mIL-13, mAMCase, mIL-33, mIFNγ, mPD-L1, hPD-L2 and hS9 were obtained from the mouse or human primer depot64 and designed to span an intronic region to avoid co-amplification of genomic DNA. Gene expression in mouse cells was analysed by real-time PCR using the iCycler system (BioRad) and SYBR green, while gene expression in human bronchial biopsies was done by real-time PCR using the StepOnePlus PCR system (Applied Biosystems) and SYBR green. Gene expression analysis was performed using the ΔΔCt method described elsewhere 65.
Bone marrow cells (3×105 cells/ml) were cultured in complete RPMI 1640 supplemented with GM-CSF (10 ng/ml, Peprotech, Rocky Hill, NJ, USA). Medium was changed on day 3 and harvested on day 6. 250,000 cells were plated on wells coated with 5 μg/ml PD-L1-Fc, PD-L2-Fc, or control IgG-Fc (R&D Systems) and stimulated with 30 μg/ml HDM or100 ng/ml LPS for 24 hours. Culture supernatants were harvested to assay IL-12 p70, IL-6, TNF-α, IL-1β (Pharmingen) and IL-23 (R&D Systems) by ELISA. Where indicated BMDCs were stimulated with IL-12, IL-13, or IL-12 + IL-13 (all 10 ng/ml) for 24 hours. RNA was harvested for PCR analysis. Where indicated cultures also included anti-PD-1 (clone RMP1-14) (eBioscience) at 5 μg/ml.
Tracheas from 4-6-week old mice were removed from the thyroid cartilage to the level of bifurcation and incubated in Pronase (1 mg/ml; Roche Applied Science, Indianapolis, IN) and incubated (18 h × 4°C while rocking). The next day, 10% FBS and 1 mg/ml DNase (Sigma-Aldrich, St. Louis, MO) were added, and the tube inverted multiple times. The trachea was discarded; cells were washed and plated onto a cell culture plate with Primaria surface treatment (BD Biosciences, Bedford MA) for 4 h to remove fibroblasts. Non-attached cells were washed, counted and plated in DMEM/F12 (50/50) containing L-glutamine (2 mM), penicillin (100 U/ml)/streptomycin (100 μg/ml) NaHCO3 (3.6 mM), FBS (5%), Cholera toxin (0.1 μg/ml), mouse EGF (0.5 ng/ml), amphotericin B (0.25 μg/ml), bovine pituitary extract (50 μg/ml), insulin-transferrin-selenium media supplement (Sigma Chemical Corp, St. Louis MO), and retinoic acid (0.1 ng/ml). mTECs were grown on collagen-coated 6 well tissue culture plates until confluent. mTECs were stimulated with nothing, IL-13 (10 ng/ml) or IL-12 + IL-13 (both at 10 ng/ml) for 24 hours. Cells were lysed with Trizol, total RNA was isolated, and cDNA synthesized to measure gene expression by RT-PCR.
For western blot analysis of STAT6 phosphorylation, BMDCs from BABL/c mice were generated as described above. After harvest, 2.5 million BMDCs were plated in complete RPMI culture medium (containing 10% fetal bovine serum (FBS)) in single wells of a 24 well plate, and allowed to adhere for 12 hours. After 12 hours, the culture medium was removed, and BMDCs were starved in RPMI containing 0.1% FBS overnight. The following morning cells were stimulated with medium alone (containing 0.1% FBS), IL-12, IL-13 or IL-12 + IL-13 (all at 10 ng/ml) for 15 minutes. Cells were immediately placed on ice, washed with ice-cold PBS and lysed with a lysis buffer containing protease inhibitors (Roche) and phosphatase inhibitor cocktails (CalBiochem). Lysates were stored at −80° until required. Total proteins (10 μg) were separated by electrophoresis on an SDS polyacrylamide gel, and transferred onto PVDF membranes and blocked with 5% BSA for 1 hr at RT. Afterwards, the membranes were incubated overnight with primary antibodies (in 5% BSA in TBS) as follows: rabbit anti-human/mouse phospho-STAT6 (Tyr761) (1:10000, Cell Signaling Technology, Beverly, MA), rabbit anti-mouse STAT6 (1:10000, Cell Signaling Technology), or anti-α-tubulin (1:10000, Cell Signaling Technology). Membranes were then washed and incubated for 1 h at RT with an HRP-conjugated goat anti-rabbit secondary (in 5% BSA in TBS at 1:10000, Cell Signaling Technology), blots were developed using an ECL Prime (GE Healthcare, Pittsburgh, PA)
To determine differences between multiple groups, analysis of variance (ANOVA) was used with post hoc comparisons using Tukey’s method. For comparison between two groups, a Student’s t-test was performed. In human samples, ANOVA was used with post hoc comparisons using the Kruskal-Wallis test. Significance was assumed at p < 0.05.
Supplementary Figure 1: Identification of pulmonary dendritic cell subsets by flow cytometry. (A) HDM-sensitized A/J mice were treated with AF405 HDM on day 14. 72 hours later mice were sacrificed and their lungs were removed. Lungs cell suspensions were stained for flow cytometric analysis of DC population. (A) Gating strategy to identify pulmonary APC populations revealed 5 distinct CD11c+ cell populations. Alveolar Macrophages (AM - CD11cbright, CD11bneg, CD317neg), plasmacytoid DCs (pDCs - CD11cdimCD11bnegCD317+), neutrophils (PMNs - CD11cbrightCD11bbrightGr1brightCD103neg), inflammatory DCs (InfDCs - CD11cbrightCD11bbrightGr1dimCD103neg) and myeloid DCs (CD11cbrightCD11bbrightGr1negCD103neg). (B) Gated DC populations were subsequently examined for expression of PD-L2, and uptake of HDM. Representative plots from a single mouse shown.
Supplementary Figure 2:In vivo PD-L2 blockade does not alter pulmonary mDC activity. A/J mice were treated with PBS, HDM, HDM + rat IgG2a control (Iso) or HDM + anti PD-L2 (αPD-L2) as described in Materials and Methods. The frequency (A), allergen content (B), and activation status of pulmonary mDCs (C) was assessed by flow cytometry. n = 8 mice from 2 independent experiments. Mean + SEM shown.
Supplementary Figure 3:In vivo PD-L2 blockade does not enhance IL-12 p40 levels in BAL. A/J mice were treated with PBS or HDM intratracheally on days 0 and 14, and 250 μg of rat IgG2a control (Iso) or anti-PD-L2 (αPD-L2) intraperitoneally on days 0, 2, 14 and 16, and mice were sacrificed on day 17. BAL was performed, and levels of IL-12 p40 were assessed by ELISA. MEAN + SEM shown. n = 10 mice from 2 independent experiments.
Supplementary Figure 4:In vivo PD-L2 or IL-12 blockade does not alter baseline tracheal pressure. A/J mice were treated with PBS or HDM intratracheally on days 0 and 14. Mice were given 250 μg of rat IgG2a control (Iso) or anti-PD-L2 (αPD-L2) intraperitoneally on days 0, 2, 14 and 16 and/or 1 mg of rat IgG2a control (Iso) or anti-IL-12 (αIL-12) intraperitoneally on days −2 and 12. Mice were sacrificed on day 17 for assessment of baseline AHR by the APTI method. Tracheal pressure was monitored for 1 minute prior to injection of intravenous acetylcholine. n = 10 mice from 2 independent experiments. Mean + SEM shown.
Supplementary Figure 5: Changes in AHR observed in mice received PD-L2 blocking and/or IL-12 blocking mAbs are not associated with changes in local changes in cytokine mRNA levels. A/J mice were treated with PBS or HDM intratracheally on days 0 and 14. Mice were given 250 μg of rat IgG2a control (Iso) or anti-PD-L2 (αPD-L2) intraperitoneally on days 0, 2, 14 and 16 and/or 1 mg of rat IgG2a control (Iso) or anti-IL-12 (αIL-12) intraperitoneally on days −2 and 12. Mice were sacrificed on day 17 and mRNA was isolated from snap-frozen lung samples for analysis of (A) IL-5 and (B) IL-13 expression by RT-PCR. n = 10 mice from 2 independent experiments. Mean + SEM shown.
Supplementary Figure 6: IL-12 and IFNγ inhibits IL-13-driven gene expression, and IL-13 inhibits IFNγ, but not IL-12-driven gene expression. (A) BMDCs were cultured in the presence of medium, IL-12, IL-13, or IL-12 + IL-13 (all at 10 ng/ml) for 18 hours. mRNA was harvested from the cells and expression of IFNγ, a representative IL-12-induced gene, was examined by RT-PCR. (B) BMDCs were cultured in the presence of medium, IFNγ, IL-13, or IFNγ + IL-13 (all at 10 ng/ml) for 18 hours. mRNA was harvested from the cells and expression of IL-13-driven genes (Fizz1, Arg1, CD206) and IFNγ-driven genes (PD-L1) was examined by RT-PCR. (C) BMDCs were cultured in the presence of IL-13, IL-12 + IL-13 (all at 10 ng/ml), or IL-12 + IL-13 + αIFNγ (at 5 μg/ml) for 18 hours. mRNA was harvested from the cells and expression of IL-13-driven genes was examined by RT-PCR. *** and * indicate p < 0.001 and p < 0.5 respectively. n = 4 replicates for each condition tested. 1 experiment of 2 performed shown.
Funded by a Parker B. Francis Fellowship, an ATS Unrestricted Research Grant, and a CCHMC Trustee Grant to IPL and NHLBI R01 HL67736-09, NIAID U19 A1070235 and NIAID R01 A1083315 to MWK
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