REVIEW

Lung transplantation: infection, inflammation,and the microbiome

Takeshi Nakajima & Vyachesav Palchevsky &

David L. Perkins & John A. Belperio & Patricia W. Finn

Received: 29 December 2010 /Accepted: 12 January 2011 /Published online: 27 January 2011# Springer-Verlag 2011

Abstract Lung transplantation is the only therapeuticoption for patients with end-stage pulmonary disorders.Despite the improvements in surgical techniques andimmunosuppressive therapy, allograft function and long-term survival are limited by the development of chroniclung transplant rejection. In this review, we focus onbronchiolitis obliterans syndrome (BOS) which is the majormanifestation of chronic lung allograft rejection. Wespecifically review the effect of infection, a risk factor forBOS, cytokines/chemokines in the pathogenesis of BOS,and the potential link between the allograft microbiome andimmune responses that may mediate the development ofBOS. Understanding the allograft microbiome and how itrelates to the pathologic mechanisms of BOS may suggesttargeted therapies to improve long-term survival post-lungtransplantation.

Keywords Lung transplantation . Bronchiolitis obliteranssyndrome . Infection . Chemokines .Microbiome

Problems of lung transplantation

Lung transplantation is a therapeutic option for end-stage lungdiseases. Lung transplant recipients (LTR) face highermortality rates and more frequent loss of graft function ascompared to other solid organ transplants, including the liver,kidney, and heart. The improvements in surgical techniques,immunosuppressive therapy, and postoperative managementhave increased the 1-year survival of LTR to approximately80%, establishing transplantation as a treatment option forend-stage lung diseases [1]. However, the 5-year survival ratehas remained approximately 50% and the 10-year graftsurvival rate is only 26% (the 2009 OPTN/SRTR AnnualReport). This low survival rate is predominately due tochronic lung allograft rejection also known as bronchiolitisobliterans syndrome (BOS) [2]. In this review, we focus onBOS which is a major manifestation of chronic lungtransplant rejection [3]. Although the precise mechanism(s)of BOS remains to be elucidated, allograft infection appearsto stimulate aberrant allo-/autoimmune responses which maydrive altered cytokine/chemokine expression, contributing tothe development of BOS. Here, we review the effect ofinfection(s) on lung transplantation as well as cytokines/chemokines which play a major role in directing alloantigen-primed T cells and other effector leukocytes into allografts,eventually causing rejection. In addition, we focus on theanalysis of the microbiome as a means of identifying knownand unknown microbes potentially associated with lungallograft rejection. We emphasize the potential link betweenthe allograft microbiome and immune responses that maymediate the development of BOS.

This article is published as part of the Special Issue on Transplantationand Tolerance.

T. Nakajima : P. W. Finn (*)Division of Pulmonary and Critical Medicine,Department of Medicine, University of California,9500 Gilman Drive 0643,La Jolla, CA 92093, USAe-mail: pwfinn@ucsd.edu

V. Palchevsky : J. A. BelperioDivision of Pulmonary, Critical Care Medicine, Allergy,and Clinical Immunology, Department of Internal Medicine,The David Geffen School of Medicine at UCLA,Los Angeles, CA, USA

D. L. PerkinsDivision of Nephrology, Department of Medicine,University of California, San Diego,La Jolla, CA, USA

Semin Immunopathol (2011) 33:135–156DOI 10.1007/s00281-011-0249-9

Bronchiolitis obliterans syndrome

Pulmonary complications after lung transplantation includehyperacute rejection, primary graft dysfunction, acuterejection, bronchial anastomotic obstruction, chronic rejec-tion, and infection. The main cause of the limited long-termsurvival is the development of chronic rejection [2]. BOS isthe clinical manifestation of chronic rejection which is afibroproliferative process that leads to an obstructiveairflow pattern and deterioration of allograft function [3].While the use of newer immunosuppressive strategiesimproves the treatment of acute rejection, there has beenno significant impact on BOS over the last 20 years. Theincidence of BOS approaches 50% within 5 years oftransplantation, and the median survival after the diagnosisof BOS is only 3 years [4]. Beyond the first year aftertransplantation, BOS accounts for over 25% of deaths. Theclinical features of BOS are progressive dyspnea and coughwith a progressive sustained decline in forced expiratoryvolume in 1 s (FEV1) that does not respond to broncho-dilators [5, 6]. The histological lesion of BOS is obliterativebronchiolitis, characterized by patchy fibroproliferation andobliteration of respiratory bronchioles, associated withperibronchiolar leukocytes, increased numbers of airwaymesenchymal cells, and increased deposition of extracellu-lar matrix [4].

The pathogenesis of BOS is complex and involves bothalloimmune and non-alloimmune mechanisms that actalone or in combination [7, 8]. Alloimmune-independentfactors which contribute to BOS are primary graft dysfunc-tion, allograft infection, and gastroesophageal reflux.Alloimmune-dependent factors, such as acute rejectionand lymphocytic bronchitis (LB), are known to besignificant risk factors for the development of BOS [5].In the early 1990s, it was recognized that peribronchiolarmononuclear cell infiltration sometimes accompaniedacute rejection, especially when the transbronchial biopsysamples were adequate. As such, LB represents airway-directed rejection. Multiple studies have identified LB asan important risk factor for chronic rejection independentof acute rejection. According to the International Societyfor Heart and Lung Transplantation definition, low-gradeLB is characterized as mononuclear cell infiltration withinthe bronchiolar submucosa without epithelial cell damageor intraepithelial mononuclear cell infiltration [9]. High-grade LB is characterized by submucosal and intraepithe-lial infiltration by mononuclear cells and eosinophils aswell as epithelial cell necrosis and metaplasia. Glanville etal. [10] identified the severity of LB as the mostsignificant risk factor for chronic rejection and anindependent risk factor for death. Acute rejection, LB,and infection(s) have consistently been identified as strongrisk factors for BOS [10–14]. Antibody-mediated rejection

may also contribute to BOS; however, further investiga-tion is needed [15]. All these risk factors for BOS augmentthe host response to the allograft in a manner similar tothat which occurs with the pulmonary host defense tomicrobes, suggesting a link between infection and lungallograft rejection.

Pulmonary host defense mechanisms

Physical barriers

The pulmonary host defense system is composed ofmultiple components, including physical barriers andimmune cells, which include epithelial cells, macrophages,neutrophils, and lymphocytes. Respiratory infection isgenerally caused by the direct invasion of microbes (e.g.,virus, bacteria, fungus). In order for pathogens to invade theupper or lower airways, they have to fight multiple physicaland immunologic barriers. The hair in the lining of thenasal track acts as a physical barrier and can potentially trapinvading organisms. The wet mucus inside the nasal cavitycan engulf microbes. The small hair-like structures (cilia)that line the lung airways constantly move any foreigninvaders up toward the pharynx to be eventually swallowedinto the stomach.

Immune responses and cytokines/chemokines

In addition to the above physical barriers, the immunesystem is important for fighting off invasion of patho-gens. The immune system defense mechanisms can bedivided into an innate (non-antigen-dependent) immuneresponse and an adaptive (antigen-dependent) immuneresponse. The innate immune response can be triggeredwhen microbes are identified by pattern recognitionreceptors, which recognize components that are con-served among broad groups of microorganisms, or whendamaged, injured, or stressed cells send out alarmsignals. The innate leukocytes, including phagocytes(macrophages, neutrophils, and dendritic cells), mastcells, eosinophils, basophils, and natural killer cells,identify and eliminate pathogens, either by contact/mediators or by engulfing and then killing microorgan-isms. Adaptive immunity, which is promoted by innateimmunity, is composed of lymphocytes, including T andB cells. The adaptive immune response requires therecognition of specific “non-self” antigens during aprocess called antigen presentation. Antigen specificityallows for the generation of responses that are tailored tospecific pathogens or pathogen-infected cells.

A delicate balance between pro- and anti-inflammatorycytokines is critical to lung wound repair from injury.

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Changes in this balance can influence lung remodeling. Thespecific mechanisms that lead to lung dysfunction mayinvolve the interactions between type 1, 2, 9, and 17 cells/immune responses. Naive cells [i.e., CD4+ T helper cells(Th), CD8+ T cytotoxic cells (Tc), mononuclear phagocytes(M), and natural killer cells (NK)] can all differentiate intomultiple distinct cell subsets, [e.g., type 1 immune cells(Th1, Tc1, M1, and NK1) and type 2 immune cells (Th2, Tc2,M2, and NK2)]; which have distinct functions and cytokineexpression profiles [16–20]. Type 1 immune response ismainly associated with the cell-mediated immune responseand is identified by the production of IL-12 and IFN-γwhich drives a cytotoxic T lymphocyte (CTL) and delayed-type hypersensitivity response. Type 2 immune response isidentified by the production of IL-4, IL-5, and IL-13 andpromotes mucosal, allergic, and humoral immunity [17–20]. Type 9 immune response is induced by IL-4 and TGF-β which augments IL-9 expression and is involved inmucus production and tissue inflammation [16]. Type 17immune response is driven by several cytokines includingTGF-β, IL-6, IL-21, and IL-23, leading to the production ofIL-6, IL-8, IL-17A, IL17F, IL-22, and IL-26, all importantin extracellular pathogen containment and a neutrophilicinflammation [16].

The nature of the antigen and the pattern of cytokinesreleased into the microenvironment are considered to be themost important factors dictating whether the immuneresponse is directed toward a type 1, 2, 9, or 17 response.In addition, during specific disease states, type 1, 2, 9, and17 immune cells can cross-regulate each other through theirrespective cytokine profiles [16, 21, 22]. These immuneresponses are important in altering lung remodeling as wellas containing and eliminating microbes.

The predominant function of chemokines is to induce themigration and activation of the leukocytes [23]. Leukocytechemotactic and activating effects of chemokines are vitalto the development of effective host responses to an antigenand the subsequent shift from innate to polarized adaptiveimmunity with the generation of CD4+ T cell subsets (e.g.,Th1, 2, 9, and 17 cells; Fig. 1).

T regulatory cells

T regulatory cells (Tregs) are a specialized subpopulation ofT cells that act to suppress the activation of the immunesystem and thereby maintain immune system homeostasisand tolerance to self-antigens. Interest in Tregs has beenheightened by evidence from experimental mouse modelsdemonstrating that the immunosuppressive potential ofthese cells can be harnessed by microbes to evadecontainment as well as their potential therapeutic use totreat autoimmune diseases and facilitate tolerance [24](Fig. 1).

Infectious susceptibility of the allograft

Despite multiple defense processes, invading microbesadapt various mechanisms to resist destruction, allowingthem to exist in symbiosis with the airway/parenchymatissue without causing disease (colonization), or producedisease by breaking through the defense barriers andinvading into the airway/parenchyma. Compared with otherorgan transplantations, LTR are more susceptible toinfections due to the direct communication of the lungwith the external environment contributing to an imbalancebetween the host’s immune system and the virulence ofmicroorganism(s).

Predisposing infectious risk factors for the allograft areits constant exposure to the external environment, surgeryvia anastomotic problems (e.g., stenosis), early airwayischemia and reperfusion injury with delayed regenerationof airway function, abnormal lymphatic drainage, anti-rejection immunosuppressive medications, spillage ofmicrobes from naïve airways and sinuses, and the devel-opment of architecture distortion of the lung allograft frominfection and rejection episodes [25].

Bronchial anastomotic problems

Anastomotic airway complications have been mainlyattributed to ischemia of the donor bronchus during theimmediate post-transplant period and occur in about onefifth of patients following lung transplantation [26, 27]. Thebronchial anastomosis is susceptible to infections in partdue to its relative devascularization after transplant,disruption of lymphatic drainage, and altered alveolarphagocytic function. Furthermore, abnormal ciliary actionin the donor epithelium, bronchial stenosis, and lack ofcough reflex due to denervation may result in microbialcolonization and subsequent complications including de-hiscence, stenosis, malacia, fistulas, granulation tissue,abscess formation, and mediastinitis.

Cystic fibrosis

Although the number of lung transplants has increasedsubstantially in the past two decades, the leading indica-tions for lung transplantation remain relatively unchanged:chronic obstructive pulmonary disease (COPD), idiopathicpulmonary fibrosis, cystic fibrosis (CF), alpha1-antitrypsindeficiency with emphysema, and pulmonary arterial hyper-tension [1]. Patients with CF have excellent survival rateswhich are similar to those with COPD after lung transplan-tation. However, they are still prone to common complica-tions including graft dysfunction and a variety of infections.Patients with CF are known to have their upper airways andsinuses colonized by Pseudomonas species, and subsequent

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downstream infection in the allograft lung is common. Inpatients receiving single lung transplants, the remainingstructurally altered native lung may harbor infection andseed the allograft [28]. The high propensity of CF patientsto re-colonization led to speculations regarding paranasalsinuses acting as a reservoir for bacterial pathogens, leadingto allograft reinfection. Patients with CF frequently havemulti-organ involvement and comorbid conditions, includ-ing malnutrition, chronic infections of the upper respiratorytract, and colonization with resistant pathogens. Coloniza-tion with multidrug-resistant Pseudomonas aeruginosa,Staphylococcus aureus, Stenotrophomonas maltophilia,and Aspergillus fumigatus are not contraindications fortransplantation because there is no evidence of effect onpost-transplant survival [29, 30]. The exception is Burkhol-deria cepacia genomovar III, which is associated with anunacceptably high risk of post-transplant mortality [31, 32].Patients with multidrug-resistant pathogens should undergofrequent evaluation for changes in their colonizing micro-flora and resistance patterns. Analysis of the microfloraguides early prophylaxis and treatment in the immediatepostoperative period. Aspergillus species are isolated beforetransplantation in 10–25% of patients with CF. Aspergilluscolonization pre-transplant can be in the form of intra-cavitary aspergilloma in patients with CF. There is lessinformation available on the significance of colonization orinfection with fungi in CF. However, subpleural cavitaryaspergillomas potentially cause empyema and decreasedpost-transplant survival. Aggressive pre- and post-transplant therapy can lead to successful transplantation[33]. Nontuberculous mycobacteria are isolated in 10–15%of CF patients prior to lung transplantation [34]. Mycobac-

terium abscessus is inherently resistant to many antibioticsand has a propensity to infect soft tissues. Individualizedapproaches are need for patients with M. abscessus whorequire lung transplantation.

Rejection

Graft rejection has historically been classified into hyper-acute rejection, acute rejection, and chronic rejection.Hyperacute rejection is a complement-mediated responsein recipients with preexisting antibodies to the donor andoccurs within minutes of reperfusion. Hyperacute rejectionis only encountered when a solid organ graft is inadver-tently transplanted across an ABO blood group barrier orwhen the recipient has been sensitized to alloantigen fromprevious exposure to blood products, past pregnancies, or afailed transplant. The clinical approach to this problem isbased on prevention through appropriate donor–recipientmatching. Acute rejection involves T cells infiltrating theallograft, predominantly in the perivascular and peribron-chiolar regions. The risk of acute rejection is highest in thefirst 3 months after transplantation. However, acute rejec-tion can also occur months to years after transplantation. Asingle episode of acute rejection is not a cause for concernif recognized and treated promptly and rarely leads to organfailure. But recurrent episodes are associated with chronicrejection. The hallmark of chronic rejection is the develop-ment of irreversible scarring, compromising the lumen ofthe small airways and the vessels. The small allograftairways develop a dense scar occluding their lumen as aresult of persistent immunologic injury. While the smallallograft airways obliterate, the large allograft airways

Fig. 1 Differentiation of CD4+ T cell subsets. Upon stimulation, naïveCD4+ T cells differentiate into five main subsets: Th1, Th2, Th9, Th17,and T regulatory cells (Tregs). IFN-γ and IL-12 induce the formation ofIFN-γ producing Th1 cells. IL-4 induces Th2 cells, producing IL-4, IL-5, and IL-13. TGF-β induces Tregs, an immune-modulating subset of

CD4+ T cells. Interestingly, in the presence of TGF-β, IL-4 induces thedifferentiation of IL-9 producing Th9 cells, and IL-6 induces Th17 cells.IL-21 and IL-23 promote the development of Th17 cells. Each subset isgenerally (but not exclusively) associated with the disease processes andpathogens listed

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develop cylindrical bronchiectasis. Such changes result inaltered airflow, decreased mucus clearances, and loss ofciliated respiratory epithelial cells. These airways are easilycolonized by gram negative rods, particularly Pseudomonas.

Immunosuppression (immunosuppressive drug)

Most lung transplant recipients are maintained on life-long pharmacologic immunosuppression, and there is asmall subset of patients who become tolerant to theirgraft. The majority of immunosuppressive regimens inclinical use exert their effect by blocking the pathwaysinvolved in the clonal expansion of alloreactive T cells orby reducing lymphocyte subpopulations. Insufficientimmunosuppression results in graft loss due to rejection,while excess immunosuppression results in increasedmorbidity and mortality from infections. The discoveryof the calcineurin inhibitor cyclosporine A (CSA)ushered in the modern era of successful solid organtransplantation. However, CSA and other current drugsused as immunosuppressants are toxic, relatively nonspe-cific regarding donor/recipient interactions, and causeproblems that contribute to morbidity and mortality aftertransplant. The complications of immunosuppressantsoccur both because of specific organ toxicity (especiallyrenal) and the increased risk of infections [35]. Immuno-suppression for lung transplantation can be consideredunder three clinical contexts: maintenance immunosup-pression, induction therapy, and anti-rejection treatment.In maintenance immunosuppression, triple-drug therapywith a calcineurin inhibitor (cyclosporine or tacrolimus),an antimetabolite (azathioprine or mycophenylate), and acorticosteroid has been the standard. Mammalian target ofrapamycin (mTOR) inhibitors that are in clinical useinclude sirolimus and everolimus. This new class of drugsacts by interfering with T cell proliferation by blocking akinase, causing cell cycle arrest [36, 37]. Early use ofmTOR inhibitors in lung recipients has been limited due toits antifibrotic effects on bronchial wound healing [38].However, the new agents have found utility in replacingthe traditional antimetabolites in triple-drug regimensbecause of their antifibrotic effects on progression ofBOS [39]. Induction agents consist of several classes ofantibodies that exhibit protective effects when adminis-tered in the peri-transplant period. Polyclonal and mono-clonal antibodies (anti-lymphocyte globulin, anti-thymocyte globulin, CD3 antibody, and IL-2 receptorantibody) have been approved for clinical use as inductionagents. Treatment of acute rejection is intensification ofimmunosuppression which is generally a “steroid pulse”consisting of 3 days of high-dose intravenous cortico-steroids, followed by a slow steroid taper back to previouslevels. Refractory acute rejection is treated with the use of

induction agents. Most clinicians attempt to both intensifyand modify the immunosuppression regimen for patientswith chronic rejection. While much clinical success hasbeen made in the treatment of acute rejection, littleprogress has been made in the treatment of chronicrejection. Although immunosuppressive therapies renderthe patients at high risk for opportunistic infection, theirbenefit for BOS is relatively-scarce. An ideal approach topreventing rejection would be to introduce a state oftolerance to the graft rather than generalized suppressionof acquired immunity. Most immunosuppressive agentsinhibit T cell activation and therefore may simultaneouslyinhibit the generation of Tregs which play an importantrole in transplant tolerance. Studies show that calcineurininhibitors diminish or abrogate Tregs activity, whereasmTOR inhibitors do not [40, 41].

Infection and BOS

Bacterial

Airway infection itself is also a reported risk factor forprogression to BOS. Bacterial bronchopulmonary infectionsare the most frequent infectious complication after lungtransplantation and comprise approximately half of allinfectious complications. In recent studies, bacterialmicrobes were isolated in up to 80% of transplant recipients[25, 42, 43]. Although antibiotic prophylaxis and a promptadjustment of the antibiotic regimen based on the isolatedmicroorganism has reduced the frequency of bacterialpneumonia, the risk of pneumonia remains marked in thelater period after lung transplantation. Late onset ofbacterial pneumonia has an association with BOS. Further-more, episodes of pulmonary infection in LTR with BOShave been associated with a faster progression through BOSstages and to death [44].

P. aeruginosa is one of the most commonly isolatedpathogen in bacterial pneumonia after lung transplanta-tion as it can be found in up to 39.4% of cases [42, 45,46]. Recipients with CF have a higher susceptibility topulmonary P. aeruginosa colonization/infection comparedwith recipients without CF [47]. Later in the time courseafter lung transplantation, the frequency of P. aeruginosacolonization in airways increases and can be associatedwith BOS, while colonization with other Gram-negativebacilli is not associated with the subsequent developmentof BOS [48]. Allograft colonization by P. aeruginosaincreases bronchoalveolar lavage fluid (BALF) IL-8 andneutrophilia and is associated with BOS. [49]. Vos et al.[50] observe in their single-center study that post-transplant P. aeruginosa colonization is an independentrisk factor for BOS and responsible for a worse survival

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with BOS. Although there is insufficient evidence of theimpact of pre-transplant P. aeruginosa colonization onpost-transplant mortality, patients with a history ofrecurrent sinusitis should be evaluated for drainage pre-transplantation.

B. cepacia complex (BCC) infection has historically beenassociated with poor outcomes after lung transplantation [51,52]. Many transplant centers consider infection with Bur-kholderia species a contraindication to lung transplantationin CF. Among BCC, Burkholderia cenocepacia (genomovarIII) and Burkholderia multivorans (genomovar II) accountfor the majority of infections. Boussaud et al. [53] recentlyreport that B. cenocepacia is associated with an increasedearly post-transplant morbidity and mortality. Alexander etal. [54] also show that B. cenocepacia infection status issignificantly associated with decreased survival at 1 yearcompared with those infected with BCC other than cenoce-pacia. Other studies show that patients infected with B.multivorans have a similar outcome to uninfected patients[55]. The role that B. cenocepacia plays in the pathogenesisof BOS remains uncertain.

Mycobacterium tuberculosis is an infrequent but impor-tant complication, afflicting 1–6% of patients [56, 57]. M.tuberculosis may be due to reactivation, occult disease inthe remaining native lung after single lung transplantation,or transmission by the transplanted lung. Identification oflatent tuberculosis infection prior to transplantation isparticularly important. Therapy is challenging due tocomplex drug interactions with immunosuppressive agents.Current guidelines recommend tuberculin skin test screen-ing in all transplant candidates followed by radiographictesting to rule out active disease.

Fungal

Fungal infection occurs in 15–35% of LTR, and over80% are caused by Candida and Aspergillus with anoverall mortality rate of nearly 60% [58–63]. WhileCandida mainly infests the upper tracheobronchial treewith only an occasional chance of dissemination, Asper-gillus has the potential to involve the deeper parenchyma.Colonization with Aspergillus species occurs in 22–85%of LTR. Invasive aspergillosis occurs in 13–26% of thecolonized LTR and is uniformly fatal. Aspergillusfumigates is the most commonly isolated species andcauses the majority of invasive diseases which may arisein the native lung after single-lung transplantation.Weight et al. [64] suggests a potential role of Aspergilluscolonization in stimulating airway inflammation leadingto the development of BOS. Significant risk factors forinvasive aspergillosis are previous cytomegalovius(CMV) disease, colonization with Aspergillus, and BOS[65–67].

Viral

Khalifah et al. [68] demonstrate that respiratory viralinfections are distinct risk factors for BOS. Bharat et al.[69] demonstrate that respiratory viruses induce suppres-sive Treg apoptosis which leads to the development ofautoimmunity and contributes to the immunopathogenesisof BOS both in humans and murine models of chronicrejection.

CMV is one of the most important and common viralinfections after lung transplantation [70]. CMV is foundin a latent stage in 60% of all adults. Routine screening ofdonor and recipient for CMV IgG allows stratification ofthose at highest risk of CMV reactivation and pneumoni-tis. Transplantation of CMV-seropositive donor lungstransmits a significant viral load to recipients. Recipientswith evidence of previous/latent CMV infection (seropos-itive recipients) are at risk, while seronegative recipientstransplanted with a seropositive pulmonary graft are athighest risk of developing CMV infection and disease afterlung transplantation. CMV infection and disease are themost relevant risk factors for the development of BOS [6].Interestingly, pulmonary CMV increases several chemo-kines associated with BOS and mortality post-lungtransplantation [71]. Strategies for prophylaxis, as wellas early diagnosis and treatment of CMV disease, may becrucial for further improvement of the results of lungtransplantation.

Adenovirus infection poses a serious problem in thepediatric LTR [72]. At least half of the patients die inrespiratory failure because of diffuse alveolar damageinduced by the virus. Occasional cases of adults withadenovirus infections are documented. In most of them, theclinical course is fatal and obliterative bronchiolitis devel-ops in the survivors [73, 74].

Epstein–Barr virus (EBV) is an oncogenic virus and isstrongly associated with post-transplant lymphoprolifer-ative disease (PTLD) [75, 76]. PTLD is believed to occurin almost half of the patients who are EBV-seronegativeand receive an EBV-seropositive graft. PTLD is thought tobe a lymphoproliferation of EBV-infected B cells arisingin the setting of over-immunosuppression. The relation-ship between active EBV infection and transplant dys-function or BOS is not clear.

Immune responses to an allograft

The CD4+ T cell plays a pivotal role in orchestrating theimmune response to an allograft. There are two pathwaysby which alloreactive T cells recognize foreign antigenand become activated: direct allorecognition and indirectallorecognition [77] (Fig. 2). The direct pathway involves

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donor antigen-presenting cells (APCs) that are carried as“passenger leukocytes” into the recipient. Direct allor-ecognition occurs when the recipient’s CD4+ T cellrecognizes intact donor major histocompatibility complex(MHC) molecules on APCs of donor origin. AlloreactiveT cells infiltrate the allograft and release cytokines/chemokines, which activate other cells (e.g., macrophages,B cells, and plasma cells) and elicit the release of growthfactors, cytotoxic mediators, and alloantibodies, all cul-minating in allograft injury [78]. Passenger leukocytes areno longer present in the allograft within weeks/monthspost-transplantation, ending the direct pathway of allor-ecognition. Thus, this recognition is more relevant to theprocess of acute rejection. Indirect allorecognition occurswhen recipient APCs processes alloantigens in theconventional manner, and the resultant peptide fragmentsare presented to recipient CD4+ T cells in the context ofself-MHC molecules. Because all of the parenchymal cellsof an allograft can serve as substrate for antigen process-ing by recipient APCs, indirect allorecognition is an everpresent mechanism in the long-term alloresponse of a hostto a graft and is important in the pathogenesis of chronicrejection [79, 80] (Fig. 2).

The role of type 1 cytokines during the pathogenesisof lung allograft dysfunction

IL-1 and TNF-α are proximal pro-inflammatory type 1cytokines that increase many pro-inflammatory molecules,including chemokines and adhesion molecules [81–86]. IL-1 and TNF-α have both been implicated in ischemia–reperfusion injury (IRI) and acute and chronic rejection inhuman studies as well as animal models of lung and airwayallograft dysfunction [87–90]. In animal models, theinhibition of either cytokine can attenuate allograft dys-function [87, 91], suggesting that current biologics avail-able to inhibit these cytokine pathways might beinformative in human lung allograft dysfunction.

IL-12 is important for the promotion of cell-mediatedtype 1 immune response and has been shown to induceIFN-γ during an allogeneic response [92–104]; however,the exact role of the IL-12 family during allograft rejectionremains controversial. Some studies suggest IL-12 causesallo-injury while others do not [92, 93, 97, 103–111]. Withregard to lung allograft dysfunction, overexpression of IL-12 accelerates airway allograft injury [112]. Similarly, in atranslation/longitudinal study of 44 human LTR, those with

Fig. 2 Direct and indirect allorecognition. Full CD4+ T cell activationrequires both a primary signal from the T cell receptor (TCR)recognition of peptide antigens and a second signal from theinteraction of co-stimulatory molecules (CD80/CD86 and CD28). Tcells recognize alloantigens by two distinct pathways known as thedirect and indirect. a Direct allorecognition occurs when therecipient’s CD4+ T cell recognizes intact donor major histocompati-bility complex (MHC) molecules on antigen-presenting cells (APCs)of donor origin. As a result, direct allorecognition between host T cells

and transplanted cells leads to sensitization, proliferation, andcytotoxic response against the graft. This recognition is relevant tothe process of acute rejection. b Indirect allorecognition occurs whenrecipient APCs processes alloantigens in the conventional manner, andthe resultant peptide fragments are presented to recipient’s CD4+ Tcells in the context of self-MHC molecules. Persistent T cellalloresponses to antigens on recipient APCs and to donor APCMHC proteins may be a major driving force for cellular death andchronic allograft rejection

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elevated levels of IL-12 in BALF were more likely todevelop BOS [113], supporting a role for IL-12 in thepathogenesis of BOS.

IFN-γ is a type 1 cytokine that can be induced by IL-12and inhibited by IL-10 [114–116]. Using a rat heterotopictracheal transplantation model of BOS, IFN-γ is persistent-ly elevated during fibro-obliterative events [117]. However,other animal models of acute and chronic rejectiondemonstrate a few unanticipated results. Using an immu-nosuppressed murine cardiac transplantation model, IFN-γ−/− recipients demonstrate accelerated acute parenchymalrejection [118–120]. Interestingly, these allografts demon-strate decreased chronic transplantation coronary arteryrejection [118–120]. Collectively, these studies suggest arole for IFN-γ, in part, by limiting acute allograft rejectiondue to anti-proliferative effects on T cells and decreasedCTL lytic activity against allogeneic targets. In contrast,IFN-γ secretion by activated infiltrating cells may, in part,worsen chronic rejection by increasing integrin and MHCclass II on the surface of allograft endothelial cells,culminating in the accumulation of activated macrophagessecreting pro-fibrotic mediators.

Pertaining to human lung transplantation, elevatedexpression of IFN-γ from BALF is associated with acuteand refractory lung allograft rejection, both risk factors forthe development of BOS [121, 122]. In addition, asignificant correlation is detected between the presence ofa high expressing human polymorphism at position +874 ofthe IFN-γ gene and BOS [123]. More animal and humanstudies will be required to determine the exact influence ofreceptor/IFN-γ biology during the continuum of early tolate lung allograft dysfunction.

IL-15 is another type 1 pro-inflammatory cytokine that ispredominately produced by activated mononuclear phag-ocytes [124–127]. Under physiological conditions, IL-15 isinvolved in the differentiation and proliferation of NK andT cells [124–132]. Increased intragraft expression of IL-15is associated with human acute rejection in both kidney andcardiac allografts [133–136]. Studies of IL-15 biology areneeded to evaluate its potential role in lung allograftdysfunction.

The role of type 2 cytokines during the pathogenesis oflung allograft dysfunction

IL-10 is a type 2 cytokine with immunomodulatorybioactivity including the inhibition of cytotoxicity and thedown-regulation of MHC class II antigens and pro-inflammatory cytokine production [116, 137–150]. Therole of IL-10 in modulating the response to allograftrejection is unclear [119, 151–160]. Nonetheless, augmen-tation of IL-10 is to be protective in multiple models oflung IRI [161–163]. Similarly, overexpression of IL-10

attenuates acute lung allograft rejection when used in a ratorthotopic single-lung transplantation model with or with-out the addition of cyclosporin [164]. Moreover, neutrali-zation of IL-10 in a rat heterotopic tracheal transplantationmodel accelerates airway obliteration, whereas the admin-istration of physiologic doses of IL-10 attenuates allograftairway fibroplasia [165–168]. With regard to human LTR,the increased IL-10 production genotype (GCC/GCC)protects recipients against acute rejection when comparedwith the intermediate or decreased IL-10 productiongenotypes [169]. The discrepancy in the effect of IL-10during allograft dysfunction may be related to dose(physiologic versus super-physiologic), type (viral versuscellular IL-10), timing of the dose, and the compartment(systemic or local administration) by which IL-10 isdelivered.

IL-13 is a potent type 2 cytokine elevated in the BALFfrom patients with BOS [170, 171]. Importantly, BALF IL-13 levels are biologically active as ex vivo experimentsdemonstrate that IL-13 induces fibroplasia [170, 171].Translational studies in rodent models of BOS demonstratethat inhibition of IL-13 biology attenuates airway allograftobliteration [170, 171]. IL-13 may be a target to investigateregarding the potential to improve long-term survival post-lung transplant.

The role of type 17 cytokines during the pathogenesis oflung allograft dysfunction

IL-6 is a cytokine with both pro-inflammatory and anti-inflammatory properties and has been associated with aTh17 profile and fibrogenesis [16, 21, 22, 172–176]. In arodent lung allograft dysfunction model, IL-6 has a bimodaldistribution (e.g., increased with IRI and then during acuterejection) [177, 178]. Similarly, elevated levels of humanIL-6 in BALF are associated with refractory acute lungallograft rejection as well as BOS [178, 179]. These studiesunderscore the possible role for IL-6 during the pathogen-esis of BOS [123].

While TGF-β has a strong history of immunosuppres-sive activity, it is the most potent inducer of collagensynthesis, fibroblast proliferation, and fibroblast chemotaxisand is recently associated with a Th17 profile [16, 180,181]. Studies involving solid organ transplantation demon-strate that TGF-β has beneficial effects during acuteallograft rejection [182–184]. Conversely, the administra-tion of adenoviral-mediated soluble TGF-β IIIR; a func-tional TGF-β antagonist, attenuates rodent BOS [185].Similar results are seen with Smad3−/− (Smad3 is asignaling pathway that mediate many of the fibrogeniceffects of TGF-β1) recipients in a rodent model of BOS[186]. Human studies involving lung transplantation showaugmented expression of TGF-β to be an early marker of

142 Semin Immunopathol (2011) 33:135–156

BOS that may correlate with the severity of luminal fibrosis[187–189]. More human studies involving TGF-β and lungtransplantation will be required to determine whether itplays a specific role in BOS.

IL-17 is a pro-inflammatory cytokine that is predomi-nately produced by T cells [190–193]. IL-17 stimulatesmultiple cell types to express IL-6, IL-1β, CXCL8, TNF-α,G-CSF, CCL2, and PGE2 [193, 194]. Excessive amounts ofIL-17 cells are thought to play a key role in autoimmunedisease. A more natural role for IL-17 is suggested bystudies which demonstrate preferential induction of IL-17in cases of host infection with various bacterial and fungalspecies. Th17 cells primarily produce two main members ofthe IL-17 family, IL-17A and IL-17F, which are involved inthe recruitment, activation, and migration of neutrophils[195, 196].

IL-17 stimulates proliferation of alloreactive T cells andthe maturation of dendritic cells (DC) [197]. Burlingham etal. [198] demonstrate that de novo autoimmunity mediatedby collagen (V)-specific Th17 cells may contribute to theprogressive airway obliteration seen in human BOS.Furthermore, in a human cross-sectional study, BALF cellpellet expressions of IL-23 and IL-17 are associated withBOS [199]. Anti-IL-23 antibody impairs the function of IL-23 in inducing IL-17 production in a rat model of airwayallograft rejection [200]. Overall, these studies suggest arole for the type 17 immune response during the pathogen-esis of BOS.

The role of receptors/chemokines during the pathogenesisof lung allograft dysfunction

The hallmark of allograft dysfunction is the infiltration ofleukocytes to the lung allograft. The ability to maintainleukocyte recruitment throughout the continuum of early(IRI/primary graft dysfunction and acute lung allograftrejection) to late (BOS) lung allograft dysfunction despiteaggressive immunosuppression is pivotal in the transitionfrom inflammation/immune responses to fibroplasia of theallograft. This persistent elicitation of mononuclear cellsrequires intercellular communication between infiltratingleukocytes, endothelium, parenchymal cells, and compo-nents of the extracellular matrix. These events are mediatedvia the generation of adhesion molecules, cytokines, andchemokines. The chemokines, by virtue of their specificcell surface receptor expression, can selectively mediate thelocal recruitment/activation of distinct leukocytes/cells,allowing for migration across the endothelium and beyondthe vascular compartment along established chemotacticgradients.

The chemokine superfamily is divided into four sub-families (C, CC, CXC, and CX3C) based on the presence ofa conserved cysteine residue at the NH2-terminus [201–

203]. CXC chemokines have been further subdivided onthe basis of the presence or absence of the sequenceglutamic acid–leucine–arginine (ELR) near the NH2-termi-nal. ELR+ chemokines are neutrophil chemoattractants withangiogenic properties. ELR-CXC chemokines are chemo-attractants of lymphocytes with angiostatic properties [204–208]. CC chemokines predominantly recruit mononuclearcells [201, 209]. The C subfamily consists of XCL1 andXCL2, which attract lymphocytes, while CX3CL1 is theonly member of the CX3C subfamily, and its domain sits ona mucin stalk allowing for cellular adhesion [210–213].

All chemokine action is mediated through seven-transmembrane-spanning G protein-coupled receptors [23].These heterotrimeric G proteins are composed of β- and -subunits. The chemokine receptors generally undergointernalization and phosphorylation following ligand bind-ing. Interaction of a ligand with its receptor leads to theexchange of GTP for GDP and the dissociation of the α-subunit from the β -subunit. The dissociated Gα and Gβ

can activate downstream signal transduction events [214,215].

Receptors/CC chemokines

In humans, elevated levels of CCL5 are associated withacute and chronic lung allograft rejection [216–218]. BothCCL3 and CCL5 are increased in an animal model of IRI,and in vivo neutralization of either chemokine reduces lunginjury [219]. In vivo depletion of CCL5 attenuates acuteand chronic lung allograft rejection in multiple animalmodels [117, 217, 218, 220, 221].

With regard to CCR2/ligand biology, CCL2 is associatedwith the continuum of human acute to chronic lungallograft rejection [220]. Rodent models of lung allograftdysfunction indicate that the inhibition of CCR2/ligandbiology attenuates IRI and acute/chronic lung allograftrejection by reducing allograft mononuclear phagocyteinfiltration [220–222].

Forkhead box P3 (FOXP3) is a gene that encodes aforkhead/winged helix transcription factor Scurfin, which isrequired for Treg development and thus is a marker of Tregcells [24, 223]. Lee et al. [224] evaluated the presence ofTregs (allograft Foxp3 expression) in tolerized (anti-CD154with donor-specific transfusion) allografts as compared tountreated allografts and as compared to isografts and naïvehearts. They found a hierarchy of Foxp3 and CCR4expression (anti-CD154 with donor-specific transfusion>>> untreated allografts >> isografts>naïve hearts). Cardiacallografts from CCR4−/− recipients treated with (anti-CD154with donor-specific transfusion therapy) have significantreductions in Foxp3 expression and reject their cardiac graftsat a normal rate. This suggests that CCR4, in this modelsystem, is critical for Treg recruitment. It will be important to

Semin Immunopathol (2011) 33:135–156 143

determine the specific detrimental and protective mecha-nisms of CCR4/ligand biology during the pathogenesis oflung allograft dysfunction.

CCR7 and its ligands CCL19 and CCL21 play a role, inpart, in the localization of antigen-loaded DC and antigen-specific T cells within T cell-rich zones, which is critical forthe initiation of an adaptive immune response [225–228].CCR7−/− BALB/c donor hearts to CCR7−/− C57BL/6recipients prolong allograft survival to 14 days and theaddition of low-dose CSA prolongs survival to 16 days[225]. Overall, CCR7 is a key regulator of DC/T cellinteraction within secondary lymphoid tissue/organs forboth the initiation of an antigen-specific T cell response andfor the initiation of alloantigen-induced cytotoxic T cellresponses. More recent human studies demonstrate thatCCR7/ligand interactions are important for Treg recruit-ment to the lung allograft associated with the prevention ofBOS [229, 230].

Meloni et al. [231] evaluates CC chemokines in BALF ofpatients in the first post-transplant year and demonstrates thatCCL19, CCL20, and CCL22 levels predict BOS onset. Weigtet al. [71] show that the alteration of BALF levels of CCL2,CCL3, and CCL5 during pulmonary CMV infection of thelung allograft predicts outcomes. Elevated levels of CCL3 inBALF are protective with regards to survival and elevatedlevels of CCL2 in BALF predicts the development of BOS,while elevated levels of CCL5 in BALF predicts an increasein mortality post-lung transplant. These studies suggest apossible utility of BALF chemokines as biomarkers forguiding risk assessment post-lung transplantation.

CX3CL1/CX3CR1

CX3CL1 is involved in direct leukocyte activation, chemo-taxis, and adhesion through its interaction with CX3CR1[210–213, 232–236]. Although there are currently nostudies of CX3CL1 and lung rejection, studies of CX3CL1show its importance during murine cardiac rejection. Inthese studies, CX3CL1 localize to the endothelium, epicar-dium, endocardium, myocardium, and infiltrating mononu-clear cells in allografts [210, 237]. In vivo antibodydepletion of CX3CL1 increases allograft survival withoutgross disruption in graft leukocyte infiltration [210].Interestingly, when CX3CR1

−/− recipients are used, thereis no effect on allograft survival, even though there is areduction in graft infiltrating NK cells [237]. The additionof CSA to the CX3CR1

−/− mice prolongs graft survival by18 days. Together, these studies suggest that NK cells of theinnate immune system and T cells of the adaptive immunesystem act synergistically to induce rejection. Also,CX3CL1 (both soluble and tethered from recipient/donorcells)/CX3CR1 interactions are important for the recruit-

ment of NK cells to the allograft. Future studies involvingCX3CL1/CX3CR1 interactions may demonstrate its impor-tance in lung allograft dysfunction.

Receptor/CXC chemokines

Interferon-inducible ELR- CXC chemokines are importantin the pathogenesis of rejection due to their chemoattractionto activated T cells [238–243]. These chemokines (CXCL9,CXCL10, and CXCL11) bind and activate through theirshared receptor, CXCR3 [205, 244, 245]. IncreasedCXCR3/ligand biology is associated with human androdent lung allograft dysfunction. Multiple experimentalmodels of allograft injury demonstrate that down-regulationof this chemokine biology inhibits allograft injury, predom-inately by reducing the recruitment of T cells and NK cellsto the allograft [121, 246–256].

Thus far, CXCL13 is a CXCR5 exclusive ligand [257].In vivo neutralization of CXCL13 leads to a ninefoldreduction of cardiac allograft infiltrating double-negative(DN) Tregs, while their homing into the spleen and lymphnodes appears to be unaffected [258]. Overall, this studydemonstrates that the CXCR5/CXCL13 biological axisplays an important role in DN Treg homing into cardiacallografts, but not secondary lymphoid organs. Overall,these data suggest that this axis may have an important rolein the generation of tolerance.

CXCR2/ligands are important in neutrophil recruitmentand angiogenesis. In human studies, CXCR2/ligands areimportant in the continuum of primary graft dysfunction toacute and chronic lung allograft rejection [187, 259–267].Similar results are found in rodent studies. The CXCR2/ligands are important for neutrophilic-dependent earlyallograft dysfunction and the vascular remodeling thatsupports BOS [259, 260].

In summary, the above human studies demonstrate theimportance of receptors/cytokines/chemokines in transplan-tation allograft dysfunction. Also, studies in animal modelsof allograft dysfunction indicate proof of principle thatreceptor/cytokine/chemokine interactions are crucial inmediating leukocyte infiltration that contribute to allograftdysfunction. Future studies of other receptors/cytokines/chemokines may foster greater understanding in thepathogenesis of lung allograft dysfunction.

The role of Tregs during the pathogenesis of lung allograftdysfunction

A decreased percentage of CD4+FOXP3+ Tregs in the BALfrom LTR is associated with the development of BOS [268].A more distinct population of Tregs (CCR7+CD3+CD4+CD25(hi)FOXP3+CD45RA− lymphocytes) is protective against the

144 Semin Immunopathol (2011) 33:135–156

development of BOS [229, 230]. Respiratory viruses induceTreg apoptosis both in humans and murine models of chronicrejection [69]. A decreased number of Tregs correlates withmore rapid onset of BOS [69]. The therapeutic application ofTregs with a potential for increased long-term survival post-lung transplantation remains to be investigated.

Microbiome

Definition

The microbiome is defined as the totality of microbes, theirgenetic elements (genomes), and environmental interactionsin a specific environment. Traditional microbiology hasfocused on the study of individual species as isolated units.However, many organisms have not been successfullycultured, identified, or otherwise characterized becausetheir growth is dependent upon a specific microenviron-ment that cannot be reproduced experimentally. Advancesin DNA sequencing technologies have created a new fieldof research, termed metagenomics, allowing comprehensiveexamination of microbial communities, even those com-prising uncultivable organisms. The metagenomic approachfacilitates comprehensive characterization of the humanmicrobiota and analysis of its role in human health anddisease.

Immune response and microbiome

Although the immune system is classically thought to haveevolved to protect from infection by microbial pathogens,humans and living organisms peacefully coexist with a vastand complex microbiota that extensively interact with theimmune system. Basic development features and functions ofthe immune system depend on interactions with the micro-biome [269]. If some bacteria are critical for shaping ahealthy immune system, the absence of these organisms maylead to disease. The impact of the microbiota on human healthis best exemplified by studies of inflammatory bowel disease(IBD), such as Crohn’s disease and ulcerative colitis [270–272]. IBD has classically been associated with increases inpro-inflammatory cytokines such as TNF-α and IFN-γ.Recently, Th17 cells and Tregs have been implicated in thepathogenesis of colitis [273–275]. Germ-free animals havedefective Th17 cell development in the small intestine, andthe reduction in IL-17 production is associated with areciprocal increase in the number of CD4+Foxp3+ Tregs[276]. Treatment of mice with colitis with probiotic bacteriaincreases the production of IL-10 and the number of Tregs[277, 278]. The concomitant reduction in intestinal inflam-mation with probiotic administration suggests a link between

a decrease in a particular species from microbiota and thedevelopment of disease

Microbiome in patients with asthma and COPD (analysisof bronchoscopic samples)

Regarding the microbiome in lung, recent studiesindicate that changes in the airway microbiome mayimpact the development of allergic inflammation andCOPD exacerbations [279, 280]. An analysis of theairway microbiota by bronchoscopic examination inpatients with asthma, COPD, and controls indicates thatthe bronchial tree is not sterile. Pathogenic proteobac-teria, particularly Haemophilus spp., are more frequent inpatients with asthma and COPD than controls. Converse-ly, Bacteroidetes, particularly Prevotella spp., are morefrequent in controls [279]. Analysis of BAL by 16SrRNA PhyloChip analysis, a culture-independent micro-array, from COPD patients who were being managed forsevere respiratory exacerbations revealed a much greaterdiversity of bacteria than has previously been appreciat-ed. Members of Pseudomonadaceae, Enterobacteria-ceae, and Helicobacteraceae are present in the airwayof COPD patients. The identification of a diverse airwaybacterial community in respiratory samples from COPDpatients suggests that the pathogenesis of these eventsmay involve a polymicrobial process [280]. The specificbacterial species remain unknown and further studies arenecessary.

Microbiome in patients with cystic fibrosis (analysis of sputumsamples)

CF is one of the most important indications for lungtransplantation, and an understanding of the microbiomeof airway infection in CF patients may improve themanagement of lung transplantation. Although only thesputum of CF patients have been studied to date, recentdata derived from molecular culture-independent analysesindicate the presence of a previously underestimatedcomplex microbial community in CF. Chronic infectionsare typically associated with a few bacterial pathogenssuch as P. aeruginosa, S. aureus, and B. cepacia complex.However, the CF airways are colonized by polymicrobialcommunities composed of a number of additional organ-isms, many of which are considered members of thenormal oropharyngeal microbiota. The recent moleculartechnologies for the accurate identification or detection ofbacteria and for the evaluation of microbial diversity haveincreased the list of emerging pathogens associated withCF patients. These recent data suggest the concept that themicrobiome ecology in CF patients is more complex and

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Fig. 3 Interactions between T helper 17 and T regulatory cells in themicrobiome of alveolar space. Alveolar epithelial cells have roles as astructural barrier and in the facilitation of mucociliary clearance inalveolar space contact with bacterial components and signals. Pro-inflammatory signals produced by pathobionts (organisms withpotential functions to induce pathology) activate innate immuneresponses and Th17 cells producing IL-17, a pro-inflammatory

cyotokine. Specific molecules produced by symbionts (organismswith known health-promoting functions) are recognized by antigen-presenting cells (APCs). This recognition activates naïve CD4+ T cellsand induces T regulatory cells (Tregs), producing IL-10, an anti-inflammatory cytokine. IL-10 suppresses the production of pro-inflammatory cytokines including IL-17

Fig. 4 Balance between T helper17 and T regulatory cells in themicrobiome. a A healthy microbiota contains a balanced compositionof symbionts, commensals, and pathobionts. Symbionts are organismswith known health-promoting functions. Commensals are thought toprovide no benefit or be a detriment to the host. Pathobionts have thepotential to induce pathology. The balance of Th17 and T regulatory

cells (Tregs) is also maintained in this situation. b Lung transplant caninduce changes in the balance between symbionts and pathobionts inthe allograft microbiota due to its infectious susceptibility, which maycause imbalance of Th17/Tregs and the development of lung allograftrejection

146 Semin Immunopathol (2011) 33:135–156

that CF may be a considered a polymicrobial infectiousdisease [281–284].

Interaction between Th17 and Tregs in the allograftmicrobiome

Interactions between the microbiome and immune re-sponse in lung transplant allografts consist of a delicatebalance due to the infectious susceptibility of allograftand the complexity of host defense response to theallograft. A model of the microbiota and interactionbetween Th17 and Tregs has been proposed [285].Signals produced by pathobionts, organisms with potentialfunctions to induce pathology, activate innate immunity aswell as Th17 cells. IL-17, a pro-inflammatory cytokine,from Th17 cells leads to injury or inflammation of tissue(Fig. 3). On the other hand, specific molecules producedby symbionts, organisms with known health-promotingfunctions, are taken up and presented to CD4+ T cells byAPCs. The recognition of the molecules by naïve CD4+ Tcells induces Tregs producing IL-10, an anti-inflammatorycytokine. IL-10 suppresses the production of IL-17 andprotects tissue damage. Whether inflammation occurs inthe lung may depend upon the responses of Th17 cells andTregs to organisms. Thus, the Th17/Tregs balance con-trolled by the microbiota is one of the most importantregulatory factors in disease. Healthy microbiota containsa balanced composition of symbionts, pathobionts, andcommensals, organisms thought to provide no benefit orbe a detriment to the host. Lung transplant can induce achange in the balance between symbionts and pathobiontsdue to multiple factors, including invasive surgery and useof antibiotics and immunosuppressant drugs. The decreaseof symbionts or increase of pathobionts may precipitateimbalance of Th17/Tregs and the development of allograftrejection (Fig. 4).

Conclusion

Despite improvements in surgical techniques and immuno-suppressive therapy, long-term outcomes after lung trans-plantation remain disappointing due to BOS. Thepathophysiology of BOS is multifactorial and complicated.Although insufficient immunosuppression results in allo-graft rejection, BOS is also strongly linked to airwayinfection caused by over-immunosuppression. Both hostimmune responses and pathogen attack need to becontrolled after lung transplant to prevent BOS. CD4+ Tcells have an important role in the immune responses to anallograft. Among CD4+ T cells, the interaction betweenTh17 cells and Tregs should be considered. Because Tregsattenuate inflammation, immunosuppressant drugs which

suppress activity of not only effector T cells but also Tregsmust be used with caution for BOS treatment or prevention.An additional caution for immunosuppression is the effecton the microbiome (the full collection of microbes thatnaturally exist in the lung). As mentioned previously,various pathogens including bacteria, fungi, and virus areinvolved in BOS. However, these pathogens constitute onlya small portion of whole microbes. New technologies allowcomprehensive examination of microbial communities andreveal previously unknown organisms. Recently, a relation-ship between the microbiome and diseases has been shown.The interaction between the changes in the microbiome andthe balance of Th17/Tregs may be closely linked to thepathogenesis of BOS. Further clarification of the micro-biome component and recognition of the changes in themicrobiome after lung transplantation are critical tounderstand the pathogenesis of allograft rejection.

Acknowledgments We thank Ko-Wei Lin PhD for a critical reviewof the manuscript. This work was supported by R01-HL077900 andR01-AI075317.

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