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Restrictive allograft syndrome (RAS) is an aggressive variant of CLAD characterized by progressive restrictive ventilatory decline and persistent pleuro-parenchymal changes that can be seen on chest CT. We identified four lung transplant recipients with a progressive restrictive ventilatory defect due to lymphocyte-predominant exudative pleural effusions, but no pleuro-parenchymal abnormalities typical of RAS. Using molecular analysis, we also found increased levels of previously described immune markers of RAS, including NFkB, 20S proteasome, lipocalin, TNFα, and TGFβ, within the circulating small extracellular vesicles of the remaining living lung transplant recipient. Despite the absence of lung parenchymal changes, these patients had a poor prognosis with rapid deterioration in allograft function and no response to pleural-based interventions such as thoracentesis, decortication, and pleurodesis. We hypothesize that these cases represent a distinct CLAD phenotype characterized by progressive restriction due to pleural inflammation, lymphocyte-predominant pleural effusion, resultant compressive atelectasis, and eventual respiratory failure in the absence of lung parenchymal involvement.

Preexisting lung-restricted autoantibodies (LRAs) are associated with a higher incidence of primary graft dysfunction (PGD), although it remains unclear whether LRAs can drive its pathogenesis. In syngeneic murine left lung transplant recipients, preexisting LRAs worsened graft dysfunction, which was evident by impaired gas exchange, increased pulmonary edema, and activation of damage-associated pathways in lung epithelial cells. LRA-mediated injury was distinct from ischemia-reperfusion injury since deletion of donor nonclassical monocytes and host neutrophils could not prevent graft dysfunction in LRA-pretreated recipients. Whole LRA IgG molecules were necessary for lung injury, which was mediated by the classical and alternative complement pathways and reversed by complement inhibition. However, deletion of Fc receptors in donor macrophages or mannose-binding lectin in recipient mice failed to rescue lung function. LRA-mediated injury was localized to the transplanted lung and dependent on IL-1β–mediated permeabilization of pulmonary vascular endothelium, which allowed extravasation of antibodies. Genetic deletion or pharmacological inhibition of IL-1R in the donor lungs prevented LRA-induced graft injury. In humans, preexisting LRAs were an independent risk factor for severe PGD and could be treated with plasmapheresis and complement blockade. We conclude that preexisting LRAs can compound ischemia-reperfusion injury to worsen PGD for which complement inhibition may be effective.

Vitamin D deficiency is prevalent in pediatric patients presenting for hematopoietic stem cell transplantation (HSCT) and has been linked to poor clinical outcomes. Using the data from a randomized control trial, in this paper we explore the effects of vitamin D supplementation on circulating cytokine levels during pediatric HSCT (www.clinicaltrials.gov as NCT03176849). A total of 41 children, 20 received Stoss therapy and 21 children received standard of care vitamin D supplementation. Levels of 25(OH)D and 20 cytokines were assessed at baseline and day +30. Significantly (P < 0.05) higher levels of mostly proinflammatory cytokines, FGF, GCSF, TNFα, IL-2, IL-6, IP10 were detected pre-transplant for patients with low compared to those with normal vitamin D levels. In sex stratified models that compare changes in cytokines between Stoss vs. standard of care, females in the Stoss group show greater changes in mostly pro -inflammatory cytokines- IP-10 (P = 0.0047), MIG (P = 0.009), and RANTES (P = 0.0047), IL-2R (P = 0.07) and IL-6(P = 0.069). Despite a small sample size, these findings suggest vitamin D deficiency affects the pre-transplant cytokine milieu and higher doses of vitamin D (Stoss therapy) appears to influence proinflammatory cytokine responses in a sex specific manner during pediatric HSCT. Larger clinical trials are warranted to validate these results.

Transplantation is a treatment option for patients diagnosed with end-stage organ diseases; however, long-term graft survival is affected by rejection of the transplanted organ by immune and nonimmune responses. Several studies have demonstrated that both acute and chronic rejection can occur after transplantation of kidney, heart, and lungs. A strong correlation has been reported between de novo synthesis of donor-specific antibodies (HLA-DSAs) and development of both acute and chronic rejection; however, some transplant recipients with chronic rejection do not have detectable HLA-DSAs. Studies of sera from such patients demonstrate that immune responses to tissue-associated antigens (TaAgs) may also play an important role in the development of chronic rejection, either alone or in combination with HLA-DSAs. The synergistic effect between HLA-DSAs and antibodies to TaAgs is being established, but the underlying mechanism is yet to be defined. We hypothesize that HLA-DSAs damage the transplanted donor organ resulting in stress and leading to the release of extracellular vesicles, which contribute to chronic rejection. These vesicles express both donor human leukocyte antigen (HLA) and non-HLA TaAgs, which can activate antigen-presenting cells and lead to immune responses and development of antibodies to both donor HLA and non-HLA tissue-associated Ags. Extracellular vesicles (EVs) are released by cells under many circumstances due to both physiological and pathological conditions. Primarily employing clinical specimens obtained from human lung transplant recipients undergoing acute or chronic rejection, our group has demonstrated that circulating extracellular vesicles display both mismatched donor HLA molecules and lung-associated Ags (collagen-V and K-alpha 1 tubulin). This review focuses on recent studies demonstrating an important role of antibodies to tissue-associated Ags in the rejection of transplanted organs, particularly chronic rejection. We will also discuss the important role of extracellular vesicles released from transplanted organs in cross-talk between alloimmunity and autoimmunity to tissue-associated Ags after solid organ transplantation.

Background. Pre–lung transplant (LTx) gastroesophageal reflux (GER) and circulating antibodies against the lung self-antigens (SAbs) collagen V and K-alpha-1 tubulin may predispose recipients to chronic lung allograft dysfunction (CLAD). We aimed to study the association of pre-LTx GER or pre-LTx SAbs with CLAD. Methods. In this retrospective analysis of patients who underwent LTx between 2015 and 2019, pre-LTx GER and SAbs were dichotomously defined as present or absent. The study group comprised recipients with either GER‚ SAbs, or both, and the control group comprised recipients without GER or SAbs. Endpoints included CLAD and survival. Results. Ninety-five LTx recipients were divided into a study group (n = 71; 75%) and a control group (n = 24; 25%). Pretransplant GER was associated with pre-LTx SAbs (odds ratio [95% confidence intervals], 5.022 [1.419-17.770]; P = 0.012). In addition, the study group (either GER‚ SAbs, or both) had a higher risk of CLAD (hazard ratio [95% confidence intervals], 8.787 [1.694-45.567]; P = 0.010) and lower CLAD-free survival after LTx than the control group (P = 0.007); however, overall survival was similar between the 2 groups (P = 0.618). Conclusions. GER was associated with elevated SAbs in LTx candidates, and either GER, SAbs, or both were associated with CLAD in LTx recipients. This association suggests that GER may cause an immune response to normally sequestered lung-associated self-antigens that drives ongoing lung injury.

Primary graft dysfunction (PGD) is the predominant cause of early graft loss following lung transplantation. We recently demonstrated that donor pulmonary intravascular nonclassical monocytes (NCM) initiate neutrophil recruitment. Simultaneously, host-origin classical monocytes (CM) permeabilize the vascular endothelium to allow neutrophil extravasation necessary for PGD. Here, we show that a CCL2-CCR2 axis is necessary for CM recruitment. Surprisingly, although intravital imaging and multichannel flow cytometry revealed that depletion of donor NCM abrogated CM recruitment, single cell RNA sequencing identified donor alveolar macrophages (AM) as predominant CCL2 secretors. Unbiased transcriptomic analysis of murine tissues combined with murine KOs and chimeras indicated that IL-1β production by donor NCM was responsible for the early activation of AM and CCL2 release. IL-1β production by NCM was NLRP3 inflammasome dependent and inhibited by treatment with a clinically approved sulphonylurea. Production of CCL2 in the donor AM occurred through IL-1R–dependent activation of the PKC and NF-κB pathway. Accordingly, we show that IL-1β–dependent paracrine interaction between donor NCM and AM leads to recruitment of recipient CM necessary for PGD. Since depletion of donor NCM, IL-1β, or IL-1R antagonism and inflammasome inhibition abrogated recruitment of CM and PGD and are feasible using FDA-approved compounds, our findings may have potential for clinical translation.

Long-term survival after lung transplant remains severely limited by chronic lung allograft dysfunction. Antibody-mediated rejection of lung transplant allografts is usually caused by donor-specific antibodies (DSAs) directed toward donor human leukocyte antigens (HLAs). Typically, patients with antibody-mediated rejection have significantly higher circulating DSAs and increased mean fluorescence intensity than those without antibody-mediated rejection. However, some patients with antibody-mediated rejection have low mean fluorescence intensities, partly due to the “sponge effect” related to DSAs binding to HLA molecules within the lung. Herein, we report the case of an 18-year-old, female lung transplant recipient who required retransplantation and developed circulating DSAs directed toward the first allograft but detected in circulation only after retransplantation. The present case draws attention to a rare finding of sponge effect in a patient with antibody-mediated rejection leading to allograft failure.

Discovery of a C3(H2O) uptake pathway has led to renewed interest in this alternative pathway triggering form of C3 in human biospecimens. Previously, a quantifiable method to measure C3(H2O), not confounded by other complement activation products, was unavailable. Herein, we describe a sensitive and specific ELISA for C3(H2O). We initially utilized this assay to determine baseline C3(H2O) levels in healthy human fluids and to define optimal sample storage and handling conditions. We detected ~500 ng/ml of C3(H2O) in fresh serum and plasma, a value substantially lower than what was predicted based on previous studies with purified C3 preparations. After a single freeze-thaw cycle, the C3(H2O) concentration increased 3- to 4-fold (~2,000 ng/ml). Subsequent freeze-thaw cycles had a lesser impact on C3(H2O) generation. Further, we found that storage of human sera or plasma samples at 4°C for up to 22 h did not generate additional C3(H2O). To determine the potential use of C3(H2O) as a biomarker, we evaluated specimens from patients with inflammatory-driven diseases. C3(H2O) concentrations were moderately increased (1.5- to 2-fold) at baseline in sera from active systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients compared to healthy controls. In addition, upon challenge with multiple freeze-thaw cycles or incubation at 22 or 37°C, C3(H2O) generation was significantly enhanced in SLE and RA patients' sera. In bronchoalveolar lavage fluid from lung-transplant recipients, we noted a substantial increase in C3(H2O) within 3 months of acute antibody-mediated rejection. In conclusion, we have established an ELISA for assessing C3(H2O) as a diagnostic and prognostic biomarker in human diseases.

Background. Ethylenediaminetetraacetic acid (EDTA) pretreatment has been shown to overcome complement interference in the single-antigen bead (SAB) assay. However, a quantitative evaluation of its impact on the assay for preemptive application to diverse clinical samples is still lacking. Methods. Serum samples from 95 renal transplant candidates were tested with and without EDTA-pretreatment in parallel. Changes in mean fluorescence intensity (MFI) values were analyzed to determine the impact of EDTA-pretreatment and the characteristics of complement interference. Results. MFI values from EDTA-treated and untreated sera showed good correlations (r = 0.99) and were linear after excluding outliers (slopes, 1; intercepts, −63.7 and −24.2 for class I and II, respectively). Using an assay cutoff of 2000 MFI, positive/negative assignments were concordant for 99% of the 9215 class I beads and 9025 class II beads tested. As defined by an MFI increment above 4000 after EDTA pretreatment, complement interference affected 172 class I beads in 12 samples (12.6%) and 60 class II beads in 7 samples (7.4%), and the findings were supported in 83% and 86% of these samples by dilution studies. In a case study, EDTA pretreatment prevented falsely low MFI values and facilitated the interpretation of titration curves. Finally, EDTA pretreatment reduced the coefficient of variance (CV) by 2.1% and 2.4% for class I and II beads respectively (P < 0.0001). Conclusions. It is safe to preemptively treat all clinical samples with EDTA before SAB assay to prevent false negative results or falsely low MFI values. EDTA pretreatment has the added benefit of improved assay precision.

Hepatitis C virus (HCV) induced liver disease is the leading indication for liver transplantation (LTx). Reinfection and accelerated development of fibrosis is a universal phenomenon following LTx. The molecular events that lead to fibrosis following HCV infection still remains poorly defined. In this study, we determined microRNA (miRNA) and mRNA expression profiles in livers from chronic HCV patients and normals using microarrays. Using Genego software and pathway finder we performed an interactive analysis to identify target genes that are modulated by miRNAs. 22 miRNAs were up regulated (>2 fold) and 35 miRNAs were down regulated (>2fold) compared to controls. Liver from HCV patients demonstrated increased expression of 306 genes (>3 fold) and reduced expression of 133 genes (>3 fold). Combinatorial analysis of the networks modulated by the miRNAs identified regulation of the phospholipase C pathway (miR200c, miR20b, and miR31through cellular proto-oncogene tyrosine-protein kinase Src (cSrc)), response to growth factors and hormones (miR141, miR107 and miR200c through peroxisome proliferator-activated receptor alpha and extracellular-signal-regulated kinases, and regulation of cellular proliferation (miR20b, miR10b, and miR141 through cyclin-dependent kinase inhibitor 1 or CDK-interacting protein 1 p21). Real time PCR (RT-PCR) validation of the miRNA in HCV infected livers demonstrated a 3.3 ±0.9 fold increase in miR200c. In vitro transfection of fibroblasts with miR200c resulted in a 2.2 fold reduction in expression of tyrosine-protein phosphatase non-receptor type 13 or FAS associated phosphatase 1 (FAP-1) and 2.3 fold increase in expression of cSrc. miR200c transfection resulted in significant increases in expression of collagen and fibroblast growth factor (2.8 and 3.4 fold, p

Liver disease due to hepatitis C virus (HCV) infection is an important health problem worldwide. HCV induced changes in microRNAs (miRNA) are shown to mediate inflammation leading to liver fibrosis. Gene expression analyses identified dysregulation of miRNA-449a in HCV patients but not in alcoholic and non-alcoholic liver diseases. By sequence analysis of the promoter for YKL40, an inflammatory marker upregulated in patients with chronic liver diseases with fibrosis, adjacent binding sites for nuclear factor of Kappa B/P65 and CCAAT/enhancer-binding protein alpha (CEBPα) were identified. P65 interacted with CEBPα to co-operatively activate YKL40 expression through sequence specific DNA binding. In vitro analysis demonstrated that tumor necrosis factor alpha (TNFα) mediated YKL40 expression is regulated by miRNA-449a and its target NOTCH1 in human hepatocytes.NOTCH1 facilitated nuclear localization of P65 in response to TNFα. Further, HCV patients demonstrated upregulation of NOTCH1 along with downregulation of miRNA-449a. Taken together it is demonstrated that miRNA-449a plays an important role in modulating expression of YKL40 through targeting the components of the NOTCH signaling pathway following HCV infection. Therefore, defining transcriptional regulatory mechanisms which control inflammatory responses and fibrosis will be important towards developing strategies to prevent hepatic fibrosis especially following HCV recurrence in liver transplant recipients.

Previous studies have shown that intrabronchial administration of antibodies (Abs) to MHC class I resulted in development of obliterative airway disease (OAD), a correlate of chronic human lung allograft rejection. Since development of Abs specific to mismatched donor HLA class II have also been associated with chronic human lung allograft rejection, we analyzed the role of Abs to MHC class II in inducing OAD. Administration of MHC class II Abs (M5/114) to C57BL/6 mice induced the classical features of OAD even though MHC class II expression is absent de novo on murine lung epithelial and endothelial cells. The induction of OAD was accompanied by enhanced cellular and humoral immune responses to self-antigens (Collagen V and K- α1Tubulin). Further, lung-infiltrating macrophages demonstrated a switch in their phenotype predominance from MΦ1 (F4/80(+)CD11c(+)) to MΦ2 (F4/80(+)CD206(+)) following administration of Abs and prior to development of OAD. Passive administration of macrophages harvested from animals with OAD but not from naïve animals induced OAD lesions. We conclude that MHC class II Abs induces a phenotype switch of lung infiltrating macrophages from MΦ1 (F4/80(+)CD11c(+)) to MΦ2 (F4/80(+)CD206(+)) resulting in the breakdown of self-tolerance along with an increase in autoimmune Th17 response leading to OAD.

The presence of a certain group of auto-antibodies (AAbs) is known to correlate with the severity of COVID-19. It is, however, unknown if such AAbs are prevalent and impact COVID-19-related outcomes in lung transplant recipients (LTRs) who are immunosuppressed. We performed a retrospective study of LTRs with COVID-19 and analyzed samples before and after COVID-19 for IgG AAbs. AAbs analysis was carried out using autoimmune and coronavirus microarray and the resulting cross-sectional differences in Ab-scores and clinical variables were analyzed using Fischer's Exact test for categorical variables and a paired t -test for continuous variables. Linear regression was used to analyze the differences in Ab-scores and COVID-19 severity. LTRs with non-severe [NS gp ( n = 10)], and severe [S gp ( n = 8)] COVID-19 disease were included. Ferritin and acute respiratory failure were higher in the S group ( p = 0.03; p < 0.0001). Among the AAbs analyzed, interferon-related AAbs (IFN-alpha2, IFN-beta, IFN lamba, IFN-epsilon), eight interleukin-related AAbs, and several tissue-related AAbs were also found to be changed significantly from pre- to post-COVID-19 ( p < 0.05). IFN-lambda ( p = 0.03) and IL-22 ( p = 0.002) were significantly associated with COVID-19 severity and remained significant in linear regression analysis while controlling for other variables. AAbs are common in LTRs, and certain groups of antibodies are particularly enhanced in LTRs with severe COVID-19. Preliminary observations of this study need to be confirmed by a larger sample size.

Background: Long-term survival after lung transplantation (LTx) is limited by chronic lung allograft dysfunction (CLAD)., Methods: We report an analysis of cytokine profiles in bronchoalveolar lavage samples collected during a prospective multicenter non-interventional trial primarily designed to determine the impact of community-acquired respiratory viral infections (CARV) in outcomes after pediatric LTx. In this analysis, we identify potential biomarkers of auto-inflammation and auto-immunity associated with survival and risk of bronchiolitis obliterans (BOS) after LTx with cytokine analysis of bronchoalveolar lavage fluid (BALF) from 61 pediatric recipients., Results: Higher IL-23 (p = .048) and IL-31 (p = .035) levels were associated with the risk of BOS, and lower levels of epithelial growth factor (EGF) (p = .041) and eotaxin (EOX) (p = .017) were associated with BOS. Analysis using conditional inference trees to evaluate cytokines at each visit associated with survival identified soluble CD30 (p < .001), pro-inflammatory cytokine IL-23 (p = .02), and sTNFRI (p = .01) below cutoff levels as associated with BOS-free survival., Conclusions: Our results indicate that post-LTx survival in children may be linked to activation of alternate pathways of the immune system that affect airway remodeling in addition to activation of "classical" pathways that have been described in adult LTx recipients. These may indicate pathways to target for intervention., (© 2022 Wiley Periodicals LLC.)

Chronic rejection predominantly manifested as bronchiolitis obliterans syndrome (BOS), still remains a major problem affecting long-term outcomes in human lung transplantation (LTx). Donor specific antibodies (DSA) and infiltration of neutrophils in the graft have been associated with the development of BOS. This study determines the role of defensins, produced by neutrophils, and its interaction with α-1-antitrypsin (AAT) towards induction of airway inflammation and fibrosis which are characteristic hallmarks of BOS. Bronchoalveolar lavage (BAL) and serum from LTx recipients, BOS+ (n=28), BOS- (n=26) and normal healthy controls (n=24) were analyzed. Our results show that BOS+ LTx recipients had higher α-defensins (HNP1-3) and β-defensin2 HBD2 concentration in BAL and serum compared to BOS-DSA-recipients and normal controls (p=0.03). BOS+ patients had significantly lower serum AAT along with higher circulating concentration of HNP-AAT complexes in BAL (p=0.05). Stimulation of primary small airway epithelial cells (SAECs) with HNPs induced expression of HBD2, adhesion molecules (ICAM and VCAM), cytokines (IL-6, IL-1β, IL-13, IL-8 and MCP-1) and growth-factor (VEGF and EGF). In contrast, anti-inflammatory cytokine, IL-10 expression decreased 2-fold (p=0.002). HNPs mediated SAEC activation was completely abrogated by AAT. In conclusion, our results demonstrates that neutrophil secretory product, α-defensins, stimulate β-defensin production by SAECs causing upregulation of pro-inflammatory and pro-fibrotic signaling molecules. Hence, chronic stimulation of airway epithelial cells by defensins can lead to inflammation and fibrosis the central events in the development of BOS following LTx., (Copyright © 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)