Citations

Abstract Recent studies suggest that the intestinal microenvironment regulate lung immune cells. Conversely, changes in the lung can modulate the gut immune system. Yet, mucosal-associated immune responses have been studied in a compartmentalized manner, as if there is no communication between different organs. Thus, the role of the gut-lung axis in mucosal immune responses is poorly understood. Using enterotoxigenic Escherichia coli (ETEC), we established a model of intestine-localized infection without translocation to any other tissue. In response to ETEC, type 2 innate lymphoid cells (ILC2s) and eosinophils accumulated in the intestinal lamina propria, even after bacterial clearance. In addition, oral ETEC transiently increased the numbers of ILC2 and eosinophils in the lung parenchyma. To test if oral ETEC promote lung immune responses to allergen, we exposed mice that recently cleared ETEC to intranasal papain/ovalbumin allergy induction model. Unexpectedly, ETEC-exposed mice were significantly resistant to allergen-induced lung inflammation, with reduced number of ILC2, Type 2 effector CD4+ T cells (Th2) and eosinophils. Paradoxically, oral ETEC-induced protection against lung allergen exposure was dependent on IL-33: IL-33KO mice developed partial lung inflammation in this model, and no protection induced by ETEC exposure was observed. Importantly, protection from allergy was achieved with ETEC heat-labile toxin (LT) oral exposure; we also found that the toxicity domain of LT is needed for this protection. Our results indicate that gastrointestinal bacterial infection mediators (toxins) negatively regulate the onset of allergic inflammation and may provide a novel immunological basis for the “hygiene hypothesis”. R01AI170649 FAPESP 2021/15185-2

Abstract Background Biomarkers that predict the patients most likely to develop progressive NTM-LD are urgently needed. We hypothesize flow cytometric (FC) detection of T cell markers after ex vivo antigen challenge in PBMC can differentiate patients with progressive vs. nonprogressive NTM-LD. We also measured the serum anti-glycopeptidolipid core IgA antibody by commercial ELISA kit and compared with FC immunoprofiling results. Methods PBMCs were isolated from patients with NTM-LD and stimulated with purified protein derivative (PPD), mycobacteria-specific peptide pools (MTB300, RD1-peptides). We have performed ELISA and FC T-cell phenotypic assays to measure diagnostic accuracy of these assays by ROC and non-parametric statistics analysis. Results A total of 29 NTM-LD patients were studied and classified into 15 non-progressive and 14 progressive NTM-LD based on radiological and clinical evaluation. Patients with progressive NTM-LD showed a statistically significantly higher frequency of PPD-specific CD3+/CD8+ IFN-γ+HLA-DR+ subsets and high diagnostic accuracy compared with non-progressors: Sensitivity = 80% specificity = 71.4% with the ROC of 0.72. There were no other statistically significant differences in other antigen-stimulated T-cell subsets but a trend was observed with HLA-DR+TNF-α+, CD25+CD134+ and CD25+PDL1+ T-cell subsets in progressors. The anti-GPL IgA levels did not show a significant difference and might not be useful in distinguishing progressive from non-progressive NTM-LD. Conclusion Our study findings suggest that PPD-specific CD8+ IFN-γ+HLA-DR+ can distinguish progressive and nonprogressive NTM-LD patients, which can identify those patients at risk of disease progression.

Abstract Aging is considered to be a major risk factor for host morbidity and mortality upon acute respiratory virus infections. To better elucidate the immune determinants of viral pathogenesis during aging, we chronological characterized lung cellular and molecular profiles following influenza virus (PR8) infection in young or aged mice till 60 days post infection. Time-course scRNAseq coupled with high dimensional flow cytometry enabled the description of changes in both immune and stromal compartment between young and aged hosts. We further zoomed into the myeloid compartment and focused on the dynamic responses of lung macrophages given their complex roles in viral pathogenesis, tissue repair and lung fibrosis. Enlarged Monocyte-derived Macrophage (MoM) population as well as decreased resident-Alveolar Macrophage (AM) population were observed in the aged mice when compared to those of the young mice. Despite the self-sustain nature of AMs during homeostasis, it has been reported that Ly6Chi monocytes can gain AM identity following severe lung damage like influenza virus infection. We found that the gain of AM identity from monocytes was abrogated in the aged hosts, which likely contributed to the age-associated impairment in lung recovery after influenzas virus infection. Additionally, trajectory analysis with scRNAseq allowed us to identify IFNα/γ signaling to be potential pathways contributing to such defect. This notion is also supported by elevated IFNγ level in BAL from the aged host during lung recovery. Our results thus have improved our understanding on the cellular and molecular mechanisms by which aged hosts exhibit severe diseases and delayed recovery after respiratory viral infection.

Background: Amiodarone is underutilized due to significant off-target toxicities. We hypothesized that targeted delivery to the heart would lead to the lowering of the dose by utilizing a cardiomyocyte-targeting peptide (CTP), a cell-penetrating peptide identified by our prior phage display work. Methods: CTP was synthesized thiolated at the N-terminus, conjugated to amiodarone via Schiff base chemistry, HPLC purified, and confirmed with MALDI/TOF. The stability of the conjugate was assessed using serial HPLCs. Guinea pigs (GP) were injected intraperitoneally daily with vehicle (7 days), amiodarone (7 days; 80 mg/kg), CTP–amiodarone (5 days; 26.3 mg/kg), or CTP (5 days; 17.8 mg/kg), after which the GPs were euthanized, and the hearts were excised and perfused on a Langendorff apparatus with Tyrode’s solution and blebbistatin (5 µM) to minimize the contractions. Voltage (RH237) and Ca2+-indicator dye (Rhod-2/AM) were injected, and fluorescence from the epicardium split and was captured by two cameras at 570–595 nm for the cytosolic Ca2+ and 610–750 nm wavelengths for the voltage. Subsequently, the hearts were paced at 250 ms with programmed stimulation to measure the changes in the conduction velocities (CV), action potential duration (APD), and Ca2+ transient durations at 90% recovery (CaTD90). mRNA was extracted from all hearts, and RNA sequencing was performed with results compared to the control hearts. Results: The CTP–amiodarone remained stable for up to 21 days at 37 °C. At ~1/15th of the dose of amiodarone, the CTP–amiodarone decreased the CV in hearts significantly compared to the control GPs (0.92 ± 0.05 vs. 1.00 ± 0.03 ms, p = 0.0007), equivalent to amiodarone alone (0.87 ± 0.08 ms, p = 0.0003). Amiodarone increased the APD (192 ± 5 ms vs. 175 ± 8 ms for vehicle, p = 0.0025), while CTP–amiodarone decreased it significantly (157 ± 16 ms, p = 0.0136), similar to CTP alone (155 ± 13 ms, p = 0.0039). Both amiodarone and CTP–amiodarone significantly decreased the calcium transients compared to the controls. CTP–amiodarone and CTP decreased the CaTD90 to an extent greater than amiodarone alone (p < 0.001). RNA-seq showed that CTP alone increased the expression of DHPR and SERCA2a, while it decreased the expression of the proinflammatory genes, NF-kappa B, TNF-α, IL-1β, and IL-6. Conclusions: Our data suggest that CTP can deliver amiodarone to cardiomyocytes at ~1/15th the total molar dose of the amiodarone needed to produce a comparable slowing of CVs. The ability of CTP to decrease the AP durations and CaTD90 may be related to its increase in the expression of Ca-handling genes, which merits further study.

Combined heart-lung transplant (HTLx) is the most durable treatment available for end-stage heart and lung failure. Many patients are unable to receive combined organs due to organ availability and allocation policies prioritizing separate heart or lung transplantation. While an average of 45 HTLxs have been performed per year in the United States half the listed patients do not receive organs. Recently, donation after circulatory death (DCD) utilizing normothermic regional perfusion (NRP) has been utilized for heart allografts with excellent results, and here, we present a case utilizing mobile NRP to procure a heart and lung block from a circulatory death donor and successful implantation for a recipient in a separate center.

Abstract Background Immune checkpoint inhibitor combined with platinum‐etoposide is the standard first‐line therapy for patients with extensive‐stage small cell lung cancer (ES‐SCLC). The phase 3 clinical trials that led to the approval of chemoimmunotherapy in ES‐SCLC excluded patients who had an Eastern Cooperative Group (ECOG) performance status (PS) of 2–3. Therefore, data on the efficacy of chemoimmunotherapy in patients with an ECOG PS of 2–3 are limited. Methods A retrospective analysis was performed on patients diagnosed with ES‐SCLC who received chemoimmunotherapy (atezolizumab or durvalumab) within the Mayo Clinic Health System between January 2016 and January 2021. The objective of this study was to compare the overall survival (OS), progression‐free survival (PFS), and best clinical response to therapy in patients with an ECOG PS of 0–1 vs. patients with an ECOG PS of 2–3 who received chemoimmunotherapy for newly diagnosed ES‐SCLC. Results In total, 82 patients were included in the study. The mean ± standard deviation age was 68.1 ± 8.3 years. Of these, 56 patients were identified with an ECOG PS of 0–1, and 26 patients were identified with an ECOG PS of 2–3. The median PFS was similar regardless of ECOG PS (5.8 months [95% CI, 4.3–6.0 months] in the ECOG PS 0–1 group vs. 4.1 months [95% CI, 3.8–6.9 months] in the ECOG PS 2–3; p = .2994). The median OS was also similar regardless of ECOG PS (10.6 months [95% CI, 8.4–13.4 months] in the ECOG PS 0–1 group vs. 9.3 months [95% CI, 4.9–12.8 months]; p = .2718) in the ECOG PS 2‐3 group. Conclusions The study results demonstrated no significant difference in PFS or OS among the ECOG PS 2–3 and ECOG PS 0–1 groups. Therefore, chemoimmunotherapy should be considered for patients who have ES‐SCLC with an ECOG PS of 2–3.

Abstract INTRODUCTION Lung cancer is the most common tumor to metastasize to the brain, as 40% of patients develop brain metastasis (BM). Once diagnosed, survival is only 6.5-10 months. A potential target to prevent BM is circulating tumor cells (CTCs). The mechanisms that mediate a CTCs adaptation to the harsh environment of circulation and BM have not been elucidated. We have demonstrated that primary lung cancer cells (LCCs) subjected to circulation have increased migration across brain endothelial cell monolayers, increased side population, and decrease survival in rodents when injected intracardially vs plated controls. METHODS We engineered a microfluidic device capable of subjecting tumor cells to prolonged circulatory shear stress (CSS) that allows the isolation/modeling of CTC-like cells. Primary LCCs (A549, H1563) were subjected to circulation, suspension, or plated (2D) for 72h as controls. At this time point, LCCs were collected, and total mRNA was extracted. Samples were submitted for RNA sequencing. Weighted gene co-expression network analysis (WCGNA) and Ingenuity Pathway Analysis were performed. (FC &gt;2 and p-value &lt;0.05). RESULTS Transcriptomic analysis indicates that cells subjected to CSS activate specific gene networks involved in cell survival, migration, invasion, adhesion of tumor cells, and cytoskeletal reorganization (z-score: &gt;2, p-value: 0.05). Canonical pathways in CTCs included ILK-signaling and Xenobiotic Metabolism. LCCs exposed to CSS had increased expression of multidrug resistance pumps ABCC2, ABCF2 (P:&lt;0.001), and side population on flow cytometry (P: &lt;0.05). The top differentially expressed genes in CTC-like cells (MMP1, ANGPTL4, and CEMIP) were involved in the invasion, angiogenesis, and pre-metastatic niche (PMN) formation of BM. CONCLUSION LCCs that survive CSS have increased expression of genes associated with migration, invasion, and chemoresistance compared to 2D controls. Targeting genes in CTCs that mediate survival and preparation of the PMN may be useful in preventing BM.

Abstract Rationale In addition to rare genetic variants and the MUC5B locus, common genetic variants contribute to idiopathic pulmonary fibrosis (IPF) risk. The predictive power of common variants outside the MUC5B locus for IPF and interstitial lung abnormalities (ILAs) is unknown. Objectives We tested the predictive value of IPF polygenic risk scores (PRSs) with and without the MUC5B region on IPF, ILA, and ILA progression. Methods We developed PRSs that included (PRS-M5B) and excluded (PRS-NO-M5B) the MUC5B region (500-kb window around rs35705950-T) using an IPF genome-wide association study. We assessed PRS associations with area under the receiver operating characteristic curve (AUC) metrics for IPF, ILA, and ILA progression. Measurements and Main Results We included 14,650 participants (1,970 IPF; 1,068 ILA) from six multi-ancestry population-based and case–control cohorts. In cases excluded from genome-wide association study, the PRS-M5B (odds ratio [OR] per SD of the score, 3.1; P = 7.1 × 10−95) and PRS-NO-M5B (OR per SD, 2.8; P = 2.5 × 10−87) were associated with IPF. Participants in the top PRS-NO-M5B quintile had ∼sevenfold odds for IPF compared with those in the first quintile. A clinical model predicted IPF (AUC, 0.61); rs35705950-T and PRS-NO-M5B demonstrated higher AUCs (0.73 and 0.7, respectively), and adding both genetic predictors to a clinical model yielded the highest performance (AUC, 0.81). The PRS-NO-M5B was associated with ILA (OR, 1.25) and ILA progression (OR, 1.16) in European ancestry participants. Conclusions A common genetic variant risk score complements the MUC5B variant to identify individuals at high risk of interstitial lung abnormalities and pulmonary fibrosis.

Abstract Rationale Global Lung Function Initiative (GLI) Global spirometry reference equations were recently derived to offer a “race-neutral” interpretation option. The impact of transitioning from the race-specific GLI-2012 to the GLI Global reference equations is unknown. Objectives Describe the direction and magnitude of changes in predicted lung function measurements in a population of diverse race and ethnicity using GLI Global in place of GLI-2012 reference equations. Methods In this multicenter cross-sectional study using a large pulmonary function laboratory database, 109,447 spirometry tests were reanalyzed using GLI Global reference equations and compared with the existing GLI-2012 standard, stratified by self-reported race and ethnicity. Measurements and Main Results Mean FEV1 and FVC percent predicted increased in the White and Northeast Asian groups and decreased in the Black, Southeast Asian, and mixed/other race groups. The prevalence of obstruction increased by 9.7% in the White group, and prevalences of possible restriction increased by 51.1% and 37.1% in the Black and Southeast Asian groups, respectively. Using GLI Global in a population with equal representation of all five race and ethnicity groups altered the interpretation category for 10.2% of spirometry tests. Subjects who self-identified as Black were the only group with a relative increase in the frequency of abnormal spirometry test results (32.9%). Conclusions The use of GLI Global reference equations will significantly impact spirometry interpretation. Although GLI Global offers an innovative approach to transition from race-specific reference equations, it is important to recognize the continued need to place these data within an appropriate clinical context.

Objective Interstitial lung disease (ILD) is an important cause of mortality in some patients with rheumatoid arthritis (RA). Patient-level factors may predict which patients with RA are at the highest risk of developing ILD and are therefore candidates for screening for this complication of the underlying disease. Methods A systematic literature review was performed using PubMed, Embase and Scopus over a 10-year period up to July 2021. Publications reporting patient-level factors in patients with RA with and without ILD that were assessed before development of ILD (or were unchanged over time and therefore could be extrapolated to before development of ILD) were retrieved for assessment of evidence. Genetic variation in MUC5B and treatment with methotrexate were not included in the assessment of evidence because these factors have already been widely investigated for association with ILD. Results We found consistent associations of age, sex, smoking status and autoantibodies with development of ILD. For biomarkers such as Krebs von den Lungen 6, which have been shown to be diagnostic for ILD, there were no publications meeting criteria for this study. Conclusions This analysis provides an initial step in the identification of patient-level factors for potential development of a risk algorithm to identify patients with RA who may be candidates for screening for ILD. The findings represent a useful basis for future research leading to an improved understanding of the disease course and improved care for patients with RA at risk of development and progression of ILD.