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Similar to IL-21 promotes pulmonary fibrosis through induction of profibrotic CD8+ T cells
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IL-21 promotes pulmonary fibrosis through induction of profibrotic CD8+ T cells
- 1. Cellular and Molecular Immunology Journal Club 01/27/2015 IL-21 promotes pulmonary fibrosis through the induction of profibrotic CD8+ T cells Tia Y. Brodeur, Tara E. Robidoux, Jason S. Weinstein, Joseph Craft, Susan L. Swain and Ann Marshak-Rothstein J Immunol. October,2015 Presented by Thi Tran-Nguyen
- 2. WHAT IS FIBROSIS? Mucosal Immunology (2009) 2, 103-121; 10.1038/mi.2008.85 Fibrotic response in normal/successful wound healing Fibrosis = unsuccessful wound healing attempt
- 3. Bleomycin induced lung fibrosis- sterile lung injury model of lung fibrosis • Chemotherapeutic antibiotic, produced by “Streptomyces verticillus”. • Used to induce experimental lung fibrosis in mice (standard protocol is via intra-tracheal route). • Cause inflammatory (IL-1, IL-6, TNF-alpha and IFN-gamma) and subsequent fibrosis reactions (TGF-beta 1, fibronectin, pro-collagen-1) in a short period of time. • The “switch” between inflammation and fibrosis occurs around day 9. • C57B1/6 mice are strong responders while BALB/C are more resistant.
- 4. Study rationale: What’s known? Th2 and Th17 play crucial roles in lung fibrosis • Th2 effector cytokines (IL-4 and IL-13) activate fibroblast • IL-13 is required to induce pulmonary fibrosis mostly via stimulating TGF-beta production by MΦ • CD4+ T cells were presumed to be the main source of IL-13 • Th17 number are found in models of fibrosis. They secrete IL- 17, but also secrete IL-21. • IL-21 can drive a progression from a Th17 to a Th2 response in fibrosing lung. • In human fibrotic lung samples, IL-21R were detected in lymphocytic infiltrate This paper seeks the role of IL-21 in pulmonary fibrosis.
- 5. IL-21 • type I cytokine (IL-2, IL-4, IL-7, IL-9, and IL-15) which shares the common cytokine receptor γ chain, γc. • produced by activated CD4+ T cells, NKT cells, and Th17 cells • has pleiotropic actions on a range of lymphoid lineages as well and non- lymphoid cells. Regulates Ig production and drives B cell terminal differentiation into plasma cells Expand CD8+ T cells and drives Th17 differentiation Has inhibitory effects on APC (DCs) Pro-apoptotic for B and NK cells Potent anti-tumor effects Implicated in the development of autoimmune diseases Curr Opin Immunol. 2008 June
- 6. IL-21- a pleotropic cytokine Curr Opin Immunol. 2008 June
- 7. IL-21R signaling is crucial for the development of lung fibrosis FIGURE 1. IL-21 promotes pulmonary fibrosis following lung injury. (B) Collagen concentrations in lung extracts Bleomycin i.t. d14 Harvest lung tissue Masson’s tri- chrome
- 8. IL-21R signaling is crucial for the development of lung fibrosis . Fig 1(C): Lung sections were stained for a-smAc to detect activated fibroblasts. Collagen in lung extracts rIL-21 d10 Harvest lung Balb/c i.t.
- 9. IL-21R signaling is crucial for the development of lung fibrosis . A small population of lung CD4+ T cells express IL-21 constitutively Sup.Fig.1.
- 10. IL-21 is upregulated in the lung in response to bleomycin induced inflammation. . Sup. 2:But IL-21R signaling is not required for bleomycin induced TGF-beta upregulation. Bleomycin i.t. d14 Collect BAL Sup. 1(C): Anti-CD3 overnight Cytokine production BAL
- 11. Fig2: IL-21R signaling is not required for lung inflammation . H&E stain
- 12. Fig3: IL-21R signaling is required for optimal T cells, especially CD8+ T cells recruitment . LNDN LNDN
- 13. Fig 3: Tregs are not responsible for the decrease in T cell recruitment to inflamed lung . LDLN Conclude: IL-21 is a key factor in maximizing CD8+ T cell responses and IL-21 has lesser effect on CD4+ T cells in fibrogenesis
- 14. Fig 4: IL-21R signaling affects IL-13, but not IL-17A production by T cells . LDLN BALF cellsLDLN
- 15. How does IL-21 affects IL-13 production by Tc2 cells? . • IL-21 could supplant IL-2 in Tc2 differentiation • Blockade of IL-2R enhances IL-13 production at the expense of IFN-g production Fig.5 In vitro stimulation of splenic CD8+ T cells
- 16. TGF-beta counteracts the effect of IL-21R signaling . IL-4/TGF-betaMedia IL-4 TGF-beta IL-21R B6 IL-21r-/- Fig.5D Surface IL-21R expression on culture CD8+ T cells
- 17. Auto-crine IL-21 amplifies Tc2 differentiation Fig.6 In vitro assay with splenic CD8+ T cells
- 18. Fig.7: IL-21 responsive CD8+ T cells are necessary and sufficient for fibrotic phenotype LDLN supernatant Lung alpha-smAC on lung sections CD21R+ T cells i.v. B6 IL-21r-/- bleomycin i.t. LDLN Anti-CD3 cytokine
- 19. Conclusion • Optimal IL-13 response to bleomycin induced lung injury requires IL-21R signaling. • IL-21R deficiency abrogate fibrosis response but does not protect from lung inflammation • IL-21R+Tc2 cells, but not Th2, can restore fibrosis in IL-21r-/- mice. • IL-21 can replace exogenous IL-2 to drive Tc2 differentiation but IL-2R blockage enhance Tc2 differentiation unique role of IL-21 that can’t be replaced by IL-2. • IL-21 is a mitogenic factor for CD8+ T cells • IL-21 differentiated Tc2 sustain their own IL-21 level.
- 20. Conclusion • Overall, the paper points to the fact that IL-21 is both necessary and sufficient for lung fibrosis -> therapeutic target • IL-21R signaling is required for optimal CD8+ T cell proliferation and recruitment. • This paper challenges the predominant view that CD4+ T cells are the driver of lung fibrosis and establish the role for IL-21 responsive Tc2 in fibrogenesis. • Tc2 serves as a link in progression from inflammation to fibrosis. • Interestingly, the study discovers that TGF-beta specifically inhibit Tc2 differentiation, which is a mechanism of fibrosis regulation.
Editor's Notes
- Figure 1 Phases of wound healing. A three-phase injury and wound-healing model describes distinct phases of a successful response. (1) Injury; many agents can cause pulmonary injury, including environmental particles, allergens, infectious agents, chemotherapy and radiation. Disruption of epithelial and endothelial cells initiate an anti-fibrinolytic cascade, temporarily plugging the affected tissue. (2) Inflammation; circulating inflammatory cells and fibrocytes are recruited to the injured site through chemokine gradients, supplying fibroblast-activating cytokines and growth factors. Neo- vascularization provides access to damaged areas and a steady stream of inflammatory, anti-inflammatory, and phagocytic cells. (3) Fibroblasts contract and decrease the size of the wound. Inflammatory cells and -SMA + myofibroblasts undergo apoptosis, terminating collagen deposition, and are cleared by phagocytic cells. Epithelial and endothelial cells are replaced and tissue architecture is restored. Fibrosis is an evolutionarily conserved process to protect against pathogens and promote wound healing. -Fibrotic scar benefits survival of species: prevent blood loss, barrier to pathogens, facilitates regeneration of damaged epithelium Fibrosis: The good and the bad Pathologic: Fibrotic diseases contribute to an estimated 45% of all-cause mortality world wide. Chronic inflammation can lead to an imbalance in the production of chemokines, cytokines, growth factors, and disrupt cellular recruitment. These changes coupled with excessive pro-fibrotic IL-13 and/or TGFbeta1 production can turn a well-controlled healing response into a pathogenic fibrotic response. Characteristics: Fibrosis results in loss of tissue architecture Progressive loss of function
- Relevant method background:
- When IL-21 is present during priming of naive CD4+ T cells under conditions leading to Th1 commitment (IL-12 plus anti-IL-4), levels of the transcription factor Eomes are down- regulated, leading to reduced production of IFN-γ. In contrast, the presence of IL-21 during priming under conditions leading to Th2 differentiation (IL-4 plus anti-IFN-γ) has no apparent effect on the production of Th2 cytokines such as IL-4. Priming of naive CD4+ T cells with TGF-β plus IL-6 leads to expression of RORγt, which initiates Th17 lineage commitment. This leads to the production of high levels of IL-21 which has the further effect of inducing the IL-23R on T cells, thus allowing Th17 cells to respond to IL-23 with further expansion and production of IL-17, IL-21, and IL-22, all of which are involved in the propagation of inflammatory responses.
- Formalin-fixed, paraffin-embedded lung sections were stained with Masson’s tri- chrome Nuclei black Cytoplasm, muscle, erythrocytes red Collagen blue
- Nuclei in blue/purple Basophils in purplish red Cytoplasm in red Muscles in dark red Erythrocytes in cherry red Collagen in pale pink Mitochondria in pale pink E) Collagen concentrations in lung extracts from PBS-, bleomycin-, IL-17A–, and IL-21–treated
- L-selectin, also known as CD62L, is a cell adhesion molecule. "homing receptor" for lymphocytes to enter secondary lymphoid tissues via high endothelial venules. Naïve TCM Effector memory T-lymphocytes do not express L-selectin, as they circulate in the periphery and have immediate effector functions upon encountering antigen.
- Sup. 1(C) B6 mice were treated with PBS or bleomycin i.t. On day 14, BAL cells were cultured with or without anti-CD3 overnight. Supernatants were collected and subjected to IL-21 ELISA.
- Nuclei in blue/purple Basophils in purplish red Cytoplasm in red Muscles in dark red Erythrocytes in cherry red Collagen in pale pink Mitochondria in pale pink
- Mice were treated i.t. with PBS or bleomycin. At day 14, LDLNs were harvested and stimulated with anti-CD3 for 24h to measure cytokine production. Most of the IL-13+ cells are CD8+. bleomycin-induced CD8+ T cell differentiation into profibrotic, type 2 effectors is highly dependent on IL-21.
- Fig.5 Purified splenic CD8+ for 4–5 d. IL-21 could supplant IL-2 in Tc2 differentiation (Fig. 5A, 5B). Remarkably, blockade of the high- affinity IL-2R, CD25, enhanced IL-13 production almost 2-fold at the expense of IFN-g production (Fig. 5B, 5C ) TGF-b–treated CD8+ T cells produced IFN-g but not IL-13 (Fig. 5B, 5C) Although TGF beta has indispensible profibrotic effects on stromal cells such as fibroblasts, it negatively regulates profibrotic CD8 T cells. => Tgf beta counter regulates effects of IL-21 signalig.
- Alone TGF beta or IL-4 alone increaases IL-21 R expression, but together they decrease IL-21R expression=> decrease CD8 T cells responsiveness to IL-21. Although TGF-b has indispensable profibrotic effects on stromal cells, such as fibroblasts, it negatively regulates profibrotic CD8+ T cells. Taken together, these data indicate that IL- 21 can supplant IL-2 to promote Tc2 differentiation during fibro- genesis and that high-affinity IL-2R signaling and TGF-b coun- terregulate the effects of IL-21 signaling.
- Fig. 6. Purified splenic CD8+ T cells from B6 mice, IL-21 reporter mice and IL-21-/- mice were cultured in the presence of indicated cytokines and Abs for 4d, washed and stimulated using anti-CD3. ere was a striking difference in IL-13 production by IL-21–deficient and IL-21–sufficient Tc2 cultures. IL-21–deficient CD8+ T cells did not secrete IL-13 when treated with IL-21 and IL-4, but wild-type B6 CD8+ T cells secreted high levels of IL-13 (Fig. 6C) C). Therefore, paracrine IL-21 drives CD8+ T cell secretion of IL-21, which, in turn, promotes IL-13 production through an autocrine pathway.
- Fig.7. Purified T cells were injected i.v. into B6 or IL-21r-/- mice who were treated with PBS or bleomycin. LDLN cells were stimulated with anti-CD3. Remark- ably, injection of B6 CD8+ T cells, but not CD4+ T cells, “res- cued” IL-13 production by LDLN T cells from bleomycin-treated Il21r2/2 recipients but had little effect on the IL-13 response of B6 recipients (Fig. 7A). Moreover, adoptive transfer of IL-21R– sufficient CD8+ T cells into Il21r2/2 mice, but not B6 mice, led to a significant increase in collagen concentration in lung extracts after bleomycin treatment, whereas adoptive transfer of IL-21R–sufficient CD4+ T cells had a negligible effect (Fig. 7B