Allograft vasculopathy (AV) leads to chronic rejection of organ transplants, but its causes are obscure. New research from the Jane-Wit laboratory showed that Sonic Hedgehog (SHH) signalling from damaged graft endothelium drives vasculopathy by promoting proinflammatory cytokine production and NLRP3-inflammasome activation in alloreactive CD4+PTCH1hiPD-1hiT memory cells, offering new diagnostic and therapeutic strategies

Crompton, Tessa

Lau, Ching-In

Rowell, Jasmine

Yánez, Diana C

Zhang, Eden

English

UCL Discovery

Trends inTIPS 2023 No. of Pages 3Pharmacological Sciences OPEN ACCESSGlossaryAllogeneic T cell response: a powerful immuneresponse that causes allograft rejection. It is aresponse against non-self and is usually polyclonal,involving interaction of the TCR with non-self MHCmolecules.C-C motif chemokine receptor 2 (CCR2): thereceptor for the chemokine CCL2 (C-C motifchemokine ligand 2), and its signalling enablescells to home to tissues that express CCL2.Cytokine signalling: signalling mediated bysecreted proteins called cytokines that signal toimmune cells and inflammatory cells to activate orregulate their functions.Inflammasome: a cellular component thatassembles in response to cellular damage ormicrobial infection, that when activated mediatescaspase-1 activation and secretion of theproinflammatory cytokines IL-1β and IL-18.Ischaemia–reperfusion injury (IRI): theexacerbation of cellular dysfunction, cell death, andtissue damage that occurs when blood flow isrestored to previously ischaemic tissues. Ischaemiaoccurs when there is a decrease in blood supply totissues, leading to decreases in oxygen and nutrients.Memory T cells (Tmem): a population of T cells thatpersists after having previously been exposed to anantigenic stimulation, such as an infection. Onre-exposure they mount a more vigourous responseto the antigen.SpotlightHedgehog signalling inallograft vasculopathy: anew therapeutic target?Jasmine Rowell,1 Ching-In Lau,1Diana C. Yánez,1Eden Zhang,1 andTessa Crompton 1,*Allograft vasculopathy (AV) leads tochronic rejection of organ trans-plants, but its causes are obscure.New research from the Jane-Wit lab-oratory showed that Sonic Hedge-hog (SHH) signalling from damagedgraft endothelium drives vasculopa-thy by promoting proinflammatorycytokine production and NLRP3-inflammasome activation inalloreactive CD4+PTCH1hiPD-1hiTmemory cells, offering new diagnos-tic and therapeutic strategies.Type I effector response: a T cell response that isdriven primarily by IFN-γ and by T helper 1 effector cells.SHH is essential during embryonic devel-opment, and as a morphogen it can sig-nal for distinct outcomes dependent onthe strength and duration of signal trans-duction [1]. After birth it regulates tissuehomeostasis and repair, and is upregu-lated in tissues following injury or induc-tion of inflammation where it signals totissue-resident and recruited immuneand inflammatory cells, and may have im-munoregulatory or proinflammatory ac-tions [2]. The canonical Hedgehog (Hh)signalling pathway is initiated by bindingof SHH – or other Hh ligands: IndianHedgehog (IHH) and Desert Hedgehog(DHH) – to their receptor, Patched1(PTCH1), thereby allowing the signaltransducer Smoothened (SMO) to bephosphorylated and enter the primary cil-ium to trigger downstream events. At theend of the pathway, the transcription fac-tors GLI1, GLI2, and GLI3 regulate ex-pression of Hh target genes through aconsensus DNA binding motif. The path-way has multiple positive and negativefeedback mechanisms, and PTCH1, itselfan Hh target gene, can function to se-quester Hh proteins. Noncanonical Hhpathway activation has also been de-scribed, including SMO-independentand PTCH1-independent signalling, andHh signals can be transduced in T cells,which lack primary cilia [1,2].AV is a major cause of chronic allograft re-jection which has been extensively studiedin heart transplant patients. In these pa-tients, cardiac allograft vasculopathy(CAV) is the leading cause of death orretransplantation in the long term, andpatients with CAV may be asymptomaticor show nonspecific symptoms, so CAVoften presents as sudden death [3].Thus, a better understanding of thecauses of AV and new strategies for its di-agnosis and prevention are urgentlyneeded. The underlying mechanism thatleads to CAV is not well understood, butit is believed to be immune-mediated,where alloreactive T cells drive a Thelper1-type (Interferon γ, IFN-γ) re-sponse to the graft endothelial cells(ECs), leading to chronic inflammation,tissue damage, and ultimately organ fail-ure [4].An exciting recent study from the Jane-Witlaboratory sheds new light on the mecha-nisms leading to AV in chronic rejection ofsolid-organ transplants, demonstrating arole for SHH secreted from damagedgraft endothelial tissue in signalling toalloreactive T cells to drive chronic vascu-lar inflammation [5].In the new study, humanised mousemodels and mixed cell cultures wereused to investigate the mechanismsthat lead to alloreactive T cell responsesagainst graft ECs [5]. The initial observa-tion that SHH expression is upregulatedin graft ECs when vascular damage oc-curs came from comparison of patientTrends in Pharmacological Sciences, Month 2023, Vol. xx, No. xx 1biopsies from renal and heart transplantrecipients who experienced delayedgraft function as a result of vasculardamage, compared with control biopsies.The impact of SHH signalling on allogeneicT cell responses (see Glossary) and vas-cular inflammation were then investigated.In vitro treatment with a SMO agonist(SAG) together with CD3 ligation, butnot SAG treatment alone, induced highcell-surface PTCH1 and programmeddeath 1 (PD-1) expression in CD4+-CD45RO+ memory T cells (Tmem)purified from peripheral blood. In cocul-ture experiments with allogeneic ECssubjected to ischaemia–reperfusioninjury (IRI), CD4+PTCH1hiPD1hiTmemcells were preferentially expanded by SHHsecreted by ECs. SHH also enhancedtype I effector responses, and expres-sion of the chemokine receptor C-Cmotif chemokine receptor 2 (CCR2),Trends in Pharmacological SciencesFigure 1. Mechanism of allograft vasculopathy (AV). The cartoon illustrates the newly describedmechanism of AV [1]. Ischaemia–reperfusion injury (IRI) leads to Sonic Hedgehog (SHH) secretion byendothelial cells which promotes type I interferon γ (IFN-γ) responses in alloreactive CD4+PTCH1hiPD1hiTmemcells. SHH activates Smoothened (SMO) signalling which leads to induction of zinc finger protein ZFYVE21.ZFYVE21 activates NLRP3 inflammasomes causing enhanced IFN-γ expression and interleukin 18 (IL-18)secretion, further driving the alloreactive response and leading to AV. The cartoon highlights novel possibletherapeutic or diagnostic strategies for AV (numbered). Abbreviations: DHH, Desert Hedgehog; IHH, IndianHedgehog; PD1, programmed death 1 protein; PTCH1, Patched1; TCR, T cell receptor. Figure created withBioRender.Trends in Pharmacological SciencesOPEN ACCESSwhich enables homing to peripheral tis-sues, indicating that this CD4+PTCH1-hiPD1hiTmem population could potentiallymigrate to the damaged graft in vivo. Theirexpansion was abrogated by pharmaco-logical inhibitors of SMO, but less so byGLI inhibitors.In vivo experiments in humanised mice –in which IRI-treated human artery seg-ments were cografted with allogeneichuman lymphocytes as a model for AV –showed that PTCH1hiTmem cells wereattenuated by treatment with pharmaco-logical SMO inhibitors or GLI inhibitors,but severity of AV and strength of thetype I response were more stronglyinhibited by SMO inhibition, and adoptivetransfer of different Tmem populations,defined by levels of cell-surface PTCH1,confirmed the contribution of thePTCH1hiTmem population in AV.Further investigation of the mechanism oftype I T cell responses showed that SAGtogether with anti-CD3 treatment in-duced the inflammasome proteinNLRP3 in PTCH1hiTmem but that thiswas an indirect effect mediated by IFN-γ, whereas in EC:T-cell cocultures phar-macological inhibition of NLRP3 reducedthe frequency of IFN-γ+PTCHhiTmem, aneffect that was rescued by exogenousIL-18. The importance of NLRP3 down-stream of SHH signalling in the allogenicT cell response was confirmed in the ani-mal model, where SAG treatment wasshown to exacerbate AV pathology, butcotreatment with an NLRP3 inhibitor to-gether with SAG reduced AV pathologyand serum IFN-γ.In the final part of the study, the T cell au-tonomous role of the inflammasome-activating zinc finger protein ZFYVE21was assessed downstream of SHH signal-ling. ZFYVE21 had previously been identi-fied as a proinflammatory protein thatleads to nuclear factor κB (NF-κB) activa-tion in ECs in vascular inflammation [6].2 Trends in Pharmacological Sciences, Month 2023, Vol. xxIn PTCH1hiTmem cells ZFYVE21 expressionwas induced in response to SAG and anti-CD3 treatment, and this induction wasmore strongly reduced by SMO inhibitionthan by GLI inhibition. Conversely, modula-tion of ZFYVE21 expression showedthat ZFYVE21 was required and sufficientfor the expansion of the PTCH1hiPD-1hiCCR2+Tmem population, but did notdirectly prime NLRP3 inflammasomes.Experiments in the AV humanised mousemodel confirmed that ZFYVE21 expressionincreased type I T cell reponses andexacerbated AV pathology to inducechronic inflammation.Taken together, this series of experi-ments contributes significantly to our un-derstanding of the mechanisms of AV,and identifies several molecules andpathways that could be targeted thera-peutically (Figure 1). Lowering SHHsignalling – either by treatment withneutralising antibodies against SHH orby pharmacological SMO inhibition –could be used to reduce the allogeneic, No. xxT cell response to damaged ECs in allo-grafts. Pharmacological SMO inhibitors arealready licensed for the treatment of somecancers, so the Hh pathway is an attractivetherapeutic target [7]. In mouse models oflung and skin inflammation, SMO inhibitionreduced SHH expression in the damagedtissue [8,9], which would have additionalbenefits for AV. Additionally, the in vivo ex-periments demonstrated that pharmaco-logical NLRP3 inhibition reduced AVpathology, and it may also have broadereffects on other inflammatory cell types.Neutralisation of IL-18 or development ofpharmacological ZFYVE21 inhibitors mightalso be effective. PD-1 agonism to inhibitthe PTCH1hiPD-1hiTmem population couldalso be considered.The identification of this new mechanism forAV may also have diagnostic benefits, aspatient blood could be screened for levelsof SHH, IL-18, and circulating PTCH1hiPD-1hiTmem; this may be particularly beneficialfor heart transplant patients in whom CAVcan develop asymptomatically.Trends in Pharmacological SciencesOPEN ACCESSSHH signalling exacerbated the T cell-driven pathology of AV and increased ex-pression of the proinflammatory cytokinesIFN-γ and IL-18, but it acted in an IFN-γ-dependent manner rather than by drivinginflammation independent of cytokinesignalling. Tissue-derived SHH signallinghas wide-ranging context-dependentpro- and anti-inflammatory effects inT cells [2]. Consistent with its role in ampli-fying the type I response in Tmem cells, ithas previously been shown to enhanceTH2 and regulatory T cell differentiation ofnaive CD4 T cells when activated in anappropriate cytokine milieu, but was un-able to drive their differentiation in theabsence of cytokine signalling [2,8,10].Given the context dependency of theoutcome of SHH signalling to T cells[2], the diverse effects of pharmacologi-cal SMO inhibition in mouse models ofinflammation [8,9], and the fact thatSHH can signal for distinct outcomesdependent on signal strength/duration,the use of SMO inhibitors for treatmentof AV in solid organ recipients will re-quire careful evaluation.AcknowledgmentsOur work is funded by the MRC (MR/S037764/1),BBSRC (BB/T020970/1), and GOSHCC (V4718).All research at UCL Great Ormond Street Instituteof Child Health is supported by the NationalInstitute for Health Research Biomedical ResearchCentre at Great Ormond Street Hospital for Chil-dren NHS Foundation Trust and University CollegeLondon.Declaration of interestsThe authors declare no financial or commercialconflicts of interest.1UCLGreatOrmondStreet Institute of Child Health, London, UK*Correspondence:t.crompton@ucl.ac.uk (T. Crompton).https://doi.org/10.1016/j.tips.2023.05.005© 2023 The Author(s). Published by Elsevier Ltd. This is an openaccess article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).Trends inReferences1. Lee, R.T. et al. (2016) Hedgehog signalling. Development143, 367–3722. Lau, C.I. et al. (2022) Sonic Hedgehog signalling in the reg-ulation of barrier tissue homeostasis and inflammation.FEBS J. 289, 8050–80613. Pober, J.S. et al. (2021) Cardiac allograft vasculopathy:current review and future research directions. Cardiovasc.Res. 117, 2624–26384. Merola, J. et al. (2017) Recent advances in allograft vascu-lopathy. Curr. Opin. Organ Transplant. 22, 1–75. Jiang, B. et al. (2023) Hedgehog-induced ZFYVE21 pro-motes chronic vascular inflammation by activating NLRP3inflammasomes in T cells. Sci. Signal. 16, eabo34066. Fang, C. et al. (2019) ZFYVE21 is a complement-inducedRab5 effector that activates non-canonical NF-kappaB viaphosphoinosotide remodeling of endosomes. Nat. Commun.10, 22477. Cortes, J.E. et al. (2019) Hedgehog signaling inhibitors insolid and hematological cancers. Cancer Treat. Rev. 76,41–508. Papaioannou, E. et al. (2019) Sonic Hedgehog signalinglimits atopic dermatitis via Gli2-driven immune regulation.J. Clin. Invest. 129, 3153–31709. Yanez, D.C. et al. (2021) Systemic pharmacologicalsmoothened inhibition reduces lung T-cell infiltration andameliorates Th2 inflammation in a mouse model of allergicairway disease. Front. Immunol. 12, 73724510. Yanez, D.C. et al. (2019) Hedgehog signaling promotes T(H)2differentiation in naive human CD4 T cells. J. Allergy Clin.Immunol. 144, 1419–1423 e1Pharmacological Sciences, Month 2023, Vol. xx, No. xx 3

Hedgehog signalling in allograft vasculopathy: a new therapeutic target?

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Hedgehog signalling in allograft vasculopathy: a new therapeutic target?

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