Primate Lung Research Articles

Abstract 1894: Preclinical development of YL205, a novel NaPi2b-targeting antibody-drug conjugate (ADC) with novel topoisomerase I inhibitor-based linker-payload for treatment of solid tumors

Abstract MediLink’s TMALIN (Tumor Microenvironment Activable LINker-payload) platform incorporates the proprietary protease-cleavable tripeptide linker and novel DNA topoisomerase I inhibitor, enables the release payload both in the tumor cell intracellularly and in the tumor microenvironment extracellularly. YL205 is built on the TMALIN platform, which is comprised of an anti-NaPi2b humanized monoclonal antibody and conjugated in a site-specific manner to yield a uniform DAR 8 product. NaPi2b, encoded by the SLC34A2 gene, is a cell surface sodium-dependent phosphate transporter that is highly expressed in certain cancers, including ovarian, lung, thyroid, and breast cancers, with limited expression in normal tissues. This study evaluated the drug properties, efficacy, safety and pharmacokinetic profiles of YL205 in nonclinical studies. YL205 achieved a high drug-to-antibody ratio through homogeneous conjugation with hydrophilic TMALIN linker-payload, which showed limited impact on the hydrophilicity of the antibody, prolonged the retention time in vivo and eventually enhanced the in vivo potency of ADC. Additionally, YL205 exhibited strong potency, high internalization, potent cytotoxicity and bystander-mediated cell killing toward tumor cells. Significant dose-dependent antitumor activities, including complete tumor regressions with no observable toxicity, were noted in NaPi2b low to high expression ovarian and NSCLC tumor xenograft mouse models. YL205 showed a favorable PK profile evidenced by the overlapping ADC and TAb curves in SD rat and cynomolgus monkey studies. Despite high levels of expression in normal lungs of non-human primates, YL205 exhibited a good safety profile with toxic findings unrelated to normal tissue target expression. Toxicity studies using cynomolgus monkeys showed that YL205 is well tolerated with calculated therapeutic index (TI, HNSTD versus MED) of ﹥80 for repeat dosing. No drug-related adverse findings were found in the lungs, liver or kidneys when tested in all doses. The highly stable nature of TMALIN linker, together with the high potency of the payload, likely mitigates the potential liability of this normal tissue expression. Based on these results, it demonstrates that YL205’s advanced ADC design results in an increased therapeutic margin, and YL205 may help address unmet medical need in patients with NaPi2b-expressing tumors. Citation Format: Liang Xiao, Wei Lian, Qigang Liu, Qing Zong, Shuai Song, Sasha Stann, Jiaqiang Cai, Tongtong Xue. Preclinical development of YL205, a novel NaPi2b-targeting antibody-drug conjugate (ADC) with novel topoisomerase I inhibitor-based linker-payload for treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1894.

Bronchioalveolar organoids: A preclinical tool to screen toxicity associated with antibody-drug conjugates

Despite extensive preclinical testing, cancer therapeutics can result in unanticipated toxicity to non-tumor tissue in patients. These toxicities may pass undetected in preclinical experiments due to modeling limitations involving poor biomimicry of 2-dimensional in vitro cell cultures and due to lack of interspecies translatability in in vivo studies. Instead, primary cells can be grown into miniature 3-dimensional structures that recapitulate morphological and functional aspects of native tissue, termed “organoids.” Here, human bronchioalveolar organoids grown from primary alveolar epithelial cells were employed to model lung epithelium and investigate off-target toxicities associated with antibody-drug conjugates (ADCs). ADCs with three different linker-payload combinations (mafodotin, vedotin, and deruxtecan) were tested in bronchioalveolar organoids generated from human, rat, and nonhuman primate lung cells. Organoids demonstrated antibody uptake and changes in viability in response to ADC exposure that model in vivo drug sensitivity. RNA sequencing identified inflammatory activation in bronchioalveolar cells in response to deruxtecan. Future studies will explore specific cell populations involved in interstitial lung disease and incorporate immune cells to the culture.

Tuberculosis in free-ranging and captive wild animals: Pathological and molecular diagnosis with histomorphological differentiation of granulomatous lesions

Tuberculosis (TB) is a serious zoonotic threat, impacting the human-livestock-wildlife interface globally. Here, we evaluated the status and histomorphological differentiation of TB lesions in 89 morbid cases of wild animals (bovids, cervids, carnivores, non-human primates, and pachyderms) in India. Histomorphological and molecular studies were done using Ziehl-Neelsen (ZN) staining, immunohistochemistry, and polymerase chain reaction (PCR), whereas cultural isolation was performed on selected samples. A total of 32 (35.95%) cases were confirmed as TB, comprising of 12 carnivores, 09 bovids, 06 cervids, 04 non-human primates, and a pachyderm. The TB lesions in the lungs, liver, and lymph nodes varied from the large-sized caseous nodules filled with dry cheesy material in bovids and cervids to variable-sized cavitations containing liquefied caseum in carnivores' lungs. The lungs, livers, and spleens of non-human primates exhibited small to medium-sized nodules. Histologically, lesions were divided into four categories (Types I, II, III, and IV) based on the extent of necrosis, the presence of mineralization, giant cells, and fibrous encapsulation. Extensive caseous necrosis with calcification, abundant giant cells, and thick fibroblastic encapsulation were consistent findings in the lungs, livers, and lymph nodes of bovids and cervids, whereas airway impaction with cellular exudate containing a teeming number of acid-fast bacilli and, at times, alveolar rupture leading to cavity formation was present in the lungs of carnivores. Absence of calcification and fibrous encapsulation was recorded in lungs of non-human primates. Immunohistochemical labelling with anti-early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) antibodies showed mild, moderate, and intense positive reactions in type II and III, type I, and type IV granulomatous lesions, respectively. Molecular detection by PCR revealed Mycobacterium tuberculosis (12 carnivores, 02 non-human primates and 01 pachyderm), M. bovis (02 cervids and 01 bovid) and M. orygis (02 cervids and 01 bovid). Cultural isolation confirmed M. tuberculosis in 03 carnivores and M. orygis in 02 cervids and 01 bovid. Our findings imply that TB is quite prevalent in the wildlife of India and there are considerable differences in the histomorphological lesions induced by distinct Mycobacterium species in different wild animals. The circulation of TB organisms in wild animals warrants a strict surveillance programme to identify the carrier status of these animals so that effective TB control strategies can be formulated to prevent spillover and spillback incidences at the human-livestock-wildlife interface.

Inhalable dry powder mRNA vaccines based on extracellular vesicles

Respiratory diseases are a global burden, with millions of deaths attributed to pulmonary illnesses and dysfunctions. Therapeutics have been developed, but they present major limitations regarding pulmonary bioavailability and product stability. To circumvent such limitations, we developed room-temperature-stable inhalable lung-derived extracellular vesicles or exosomes (Lung-Exos) as mRNA and protein drug carriers. Compared with standard synthetic nanoparticle liposomes (Lipos), Lung-Exos exhibited superior distribution to the bronchioles and parenchyma and are deliverable to the lungs of rodents and nonhuman primates (NHPs) by dry powder inhalation. In a vaccine application, severe acute respiratory coronavirus 2 (SARS-CoV-2) spike (S) protein encoding mRNA-loaded Lung-Exos (S-Exos) elicited greater immunoglobulin G (IgG) and secretory IgA (SIgA) responses than its loaded liposome (S-Lipo) counterpart. Importantly, S-Exos remained functional at room-temperature storage for one month. Our results suggest that extracellular vesicles can serve as an inhaled mRNA drug-delivery system that is superior to synthetic liposomes.

Single-cell transcriptomic profiling of lung endothelial cells identifies dynamic inflammatory and regenerative subpopulations.

Studies have demonstrated the phenotypic heterogeneity of vascular endothelial cells (ECs) within a vascular bed; however, little is known about how distinct endothelial subpopulations in a particular organ respond to an inflammatory stimulus. We performed single-cell RNA-Seq of 35,973 lung ECs obtained during baseline as well as postinjury time points after inflammatory lung injury induced by LPS. Seurat clustering and gene expression pathway analysis identified 2 major subpopulations in the lung microvascular endothelium, a subpopulation enriched for expression of immune response genes such as MHC genes (immuneEC) and another defined by increased expression of vascular development genes such as Sox17 (devEC). The presence of immuneEC and devEC subpopulations was also observed in nonhuman primate lungs infected with SARS-CoV-2 and murine lungs infected with H1N1 influenza virus. After the peak of inflammatory injury, we observed the emergence of a proliferative lung EC subpopulation. Overexpression of Sox17 prevented inflammatory activation in ECs. Thus, there appeared to be a "division of labor" within the lung microvascular endothelium in which some ECs showed propensity for inflammatory signaling and others for endothelial regeneration. These results provide underpinnings for the development of targeted therapies to limit inflammatory lung injury and promote regeneration.

An Investigational Inhaled rhIL-1Ra (ALTA-2530) Demonstrates Distribution to Distal Regions of Lung and High Affinity IL-1 Receptor Blockade Supporting Development as a Treatment for Bronchiolitis Obliterans Syndrome

<h3>Purpose</h3> Dysregulated expression of interleukin-1 (IL-1), and downstream cytokines including IL-6, has been implicated in the development and progression of bronchiolitis obliterans syndrome (BOS). Quiescence of IL-1 signaling via blockade of the IL-1 receptor type 1 (IL-1R1) is proposed to restore physiologic immune regulation. Herein, we characterize ALTA-2530: i) distribution in rat and non-human primate (NHP) lung tissue following aerosolized administration, ii) binding affinity to IL-1R1, and iii) <i>in vitro</i> suppression of IL-6 release to assess potency of receptor blockade and development of an exposure response relationship to guide dose selection for <i>in vivo</i> studies. <h3>Methods</h3> Bronchoalveolar lavage fluid (BALF) and lung tissue samples were collected from rat and NHP following 7 daily inhaled doses of ALTA-2530. ALTA-2530 was determined in BALF by affinity capture LC-MS/MS. Tissues were processed for immunohistochemistry to identify the distribution of rhIL-1Ra. Binding kinetics of rhIL-1Ra to human IL-1R1 were determined by surface plasmon resonance and compared to IL-1α and β. ALTA-2530 mediated inhibition of IL-6 release in human and NHP whole blood was determined following stimulation by IL-1β. <h3>Results</h3> Once daily administration of ALTA-2530 provided >24hr exposure in BALF. Immunohistochemistry of lungs from healthy rats and NHPs 24hr post dose demonstrated dose-dependent delivery of ALTA-2530 to alveoli and bronchial epithelial cells. Early data also indicate association with alveolar macrophages. Distribution to narrow rat bronchioles is supportive of therapeutically relevant distribution in human bronchioles - a target for the treatment of BOS. ALTA-2530 rhIL-1Ra bound to IL-1R1 with over 100X greater affinity (K_D∼10⁻¹² M, k_d∼10⁻⁵ s⁻¹, k_a∼10⁶ M⁻¹s⁻¹) than endogenous IL-1 agonists IL-1α (K_D∼10⁻⁷ M, k_d∼10⁻³ s⁻¹, k_a∼10³ M⁻¹s⁻¹) and IL-1β (K_D∼10⁻¹⁰ M, k_d∼10⁻³ s⁻¹, k_a∼10⁷ M⁻¹s⁻¹). Functional potency for IL-1R1 blockade in whole blood demonstrated ALTA-2530 rhIL-1Ra potently inhibited IL-6 release following IL-1β stimulation (human IC₅₀ 5 ng/mL; NHP IC₅₀ 7 ng/mL). <h3>Conclusion</h3> Inhaled ALTA-2530 distributes to distal regions of lung and inhibits IL-6 release supportive of a therapy for IL-1 and IL-6 driven pathologies including BOS.

Inflammatory blockade prevents injury to the developing pulmonary gas exchange surface in preterm primates.

Perinatal inflammatory stress is associated with early life morbidity and lifelong consequences for pulmonary health. Chorioamnionitis, an inflammatory condition affecting the placenta and fluid surrounding the developing fetus, affects 25 to 40% of preterm births. Severe chorioamnionitis with preterm birth is associated with significantly increased risk of pulmonary disease and secondary infections in childhood, suggesting that fetal inflammation may markedly alter the development of the lung. Here, we used intra-amniotic lipopolysaccharide (LPS) challenge to induce experimental chorioamnionitis in a prenatal rhesus macaque (Macaca mulatta) model that mirrors structural and temporal aspects of human lung development. Inflammatory injury directly disrupted the developing gas exchange surface of the primate lung, with extensive damage to alveolar structure, particularly the close association and coordinated differentiation of alveolar type 1 pneumocytes and specialized alveolar capillary endothelium. Single-cell RNA sequencing analysis defined a multicellular alveolar signaling niche driving alveologenesis that was extensively disrupted by perinatal inflammation, leading to a loss of gas exchange surface and alveolar simplification, with notable resemblance to chronic lung disease in newborns. Blockade of the inflammatory cytokines interleukin-1β and tumor necrosis factor-α ameliorated LPS-induced inflammatory lung injury by blunting stromal responses to inflammation and modulating innate immune activation in myeloid cells, restoring structural integrity and key signaling networks in the developing alveolus. These data provide new insight into the pathophysiology of developmental lung injury and suggest that modulating inflammation is a promising therapeutic approach to prevent fetal consequences of chorioamnionitis.

Human distal lung maps and lineage hierarchies reveal a bipotent progenitor.

Mapping the spatial distribution and molecular identity of constituent cells is essential for understanding tissue dynamics in health and disease. We lack a comprehensive map of human distal airways, including the terminal and respiratory bronchioles (TRBs), which are implicated in respiratory diseases1-4. Here, using spatial transcriptomics and single-cell profiling of microdissected distal airways, we identify molecularly distinct TRB cell types that have not-to our knowledge-been previously characterized. These include airway-associated LGR5+ fibroblasts and TRB-specific alveolar type-0 (AT0) cells and TRB secretory cells (TRB-SCs). Connectome maps and organoid-based co-cultures reveal that LGR5+ fibroblasts form a signalling hub in the airway niche. AT0 cells and TRB-SCs are conserved in primates and emerge dynamically during human lung development. Using a non-human primate model of lung injury, together with human organoids and tissue specimens, we show that alveolar type-2 cells in regenerating lungs transiently acquire an AT0 state from which they can differentiate into either alveolar type-1 cells or TRB-SCs. This differentiation programme is distinct from that identified in the mouse lung5-7. Our study also reveals mechanisms that drive the differentiation of the bipotent AT0 cell state into normal or pathological states. In sum, our findings revise human lung cell maps and lineage trajectories, and implicate an epithelial transitional state in primate lung regeneration and disease.

Histologic pulmonary lesions of SARS-CoV-2 in 4 nonhuman primate species: An institutional comparative review.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an emergent, amphixenotic infection that resulted in a pandemic declaration in March 2020. A rapid search for appropriate animal models of this newly emergent viral respiratory disease focused initially on traditional nonhuman primate research species. Nonhuman primate models have previously been shown to be valuable in evaluation of emerging respiratory coronaviruses with pandemic potential (ie, SARS-CoV and Middle East respiratory syndrome coronavirus). In this article, we review the pulmonary histopathologic characteristics and immunohistochemical evaluation of experimental SARS-CoV-2 infection in the rhesus macaque, pigtail macaque, African green monkey, and squirrel monkey. Our results indicate that all evaluated nonhuman primate species developed variably severe histopathologic changes typical of coronavirus respiratory disease characterized by interstitial pneumonia with or without syncytial cell formation, alveolar fibrin, and pulmonary edema that progressed to type II pneumocyte hyperplasia. Lesion distribution was multifocal, frequently subpleural, and often more severe in lower lung lobes. However, squirrel monkeys showed the least severe and least consistent lesions of the evaluated nonhuman primates. Additionally, our results highlight the disparate physical relationship between viral antigen and foci of pulmonary lesions. While classic respiratory coronaviral lesions were observed in the lungs of all nonhuman primates evaluated, none of the primates exhibited severe lesions or evidence of diffuse alveolar damage and therefore are unlikely to represent the severe form of SARS-CoV-2 infection observed in fatal human cases.

AEOL 10150 Alleviates Radiation-induced Innate Immune Responses in Non-human Primate Lung Tissue.

To study the molecular and cellular mechanisms of radiation-induced lung injury (RILI) in a non-human primate model, Rhesus macaques were irradiated with lethal doses of radiation to the whole thorax. A subset of the irradiated animals was treated with AEOL 10150, a potent catalytic scavenger of reactive oxygen and nitrogen species. Lung tissues were collected at necropsy for molecular and immunohistochemical (IHC) studies. Microarray expression profiling in the irradiated lung tissues identified differentially expressed genes (DEGs) and pathways important in innate immunity. The elevated expression of cytokines (CCL2, CCL11, IL-8), complement factors (CFB, C3), apoptosis-related molecules (p53, PTEN, Bax, p21, MDM2, c-Caspase 3), and adhesion molecules (fibronectin, integrin β6, ICAM-1) were further studied using real-time PCR, Western blot, or IHC. Oxidative stress and pulmonary inflammatory cell infiltration were increased in the irradiated lungs. Treatment with AEOL 10150 significantly decreased oxidative stress and monocyte/macrophage infiltration. Cytokine/chemokine-induced excessive innate immune response after thoracic irradiation plays an important role in RILI. To our knowledge, this is the first study to highlight the role of cytokine/chemokine-induced innate immune responses in radiation-induced pulmonary toxicity in a NHP model.

Acute Proteomic Changes in Lung after Radiation: Toward Identifying Initiating Events of Delayed Effects of Acute Radiation Exposure in Non-human Primate after Partial Body Irradiation with Minimal Bone Marrow Sparing.

Radiation-induced lung injury is a delayed effect of acute radiation exposure resulting in pulmonary pneumonitis and fibrosis. Molecular mechanisms that lead to radiation-induced lung injury remain incompletely understood. Using a non-human primate model of partial body irradiation with minimal bone marrow sparing, lung was analyzed from animals irradiated with 12 Gy at timepoints every 4 d up to 21 d after irradiation and compared to non-irradiated (sham) controls. Tryptic digests of lung tissues were analyzed by liquid chromatography-tandem mass spectrometry followed by pathway analysis. Out of the 3,101 unique proteins that were identified, we found that 252 proteins showed significant and consistent responses across at least three time points post-irradiation, of which 215 proteins showed strong up-regulation while 37 proteins showed down-regulation. Canonical pathways affected by irradiation, changes in proteins that serve as upstream regulators, and proteins involved in key processes including inflammation, fibrosis, and retinoic acid signaling were identified. The proteomic profiling of lung conducted here represents an untargeted systems biology approach to identify acute molecular events in the non-human primate lung that could potentially be initiating events for radiation-induced lung injury.

Characterization of the Onset, Progression, and Reversibility of Morphological Changes in Mouse Lung after Pharmacological Inhibition of Leucine-Rich Kinase 2 Kinase Activity.

Gain-of-function mutations in leucine-rich kinase 2 (LRRK2) are associated with increased incidence of Parkinson disease (PD); thus, pharmacological inhibition of LRRK2 kinase activity is postulated as a disease-modifying treatment of PD. Histomorphological changes in lungs of nonhuman primates (NHPs) treated with small-molecule LRRK2 kinase inhibitors have brought the safety of this treatment approach into question. Although it remains unclear how LRRK2 kinase inhibition affects the lung, continued studies in NHPs prove to be both cost- and resource-prohibitive. To develop a tractable alternative animal model platform, we dosed male mice in-diet with the potent, highly selective LRRK2 kinase inhibitor MLi-2 and induced histomorphological changes in lung within 1 week. Oral bolus dosing of MLi-2 at a frequency modeled to provide steady-state exposure equivalent to that achieved with in-diet dosing induced type II pneumocyte vacuolation, suggesting pulmonary changes require sustained LRRK2 kinase inhibition. Treating mice with MLi-2 in-diet for up to 6 months resulted in type II pneumocyte vacuolation that progressed only modestly over time and was fully reversible after withdrawal of MLi-2. Immunohistochemical analysis of lung revealed a significant increase in prosurfactant protein C staining within type II pneumocytes. In the present study, we demonstrated the kinetics for onset, progression, and rapid reversibility of chronic LRRK2 kinase inhibitor effects on lung histomorphology in rodents and provide further evidence for the derisking of safety and tolerability concerns for chronic LRRK2 kinase inhibition in PD. SIGNIFICANCE STATEMENT: We have defined a mouse model by which the on-target lung effects of leucine-rich kinase 2 (LRRK2) kinase inhibition can be monitored, whereas previous in vivo testing relied solely on nonhuman primates. Data serve to derisk long-term treatment with LRRK2 kinase inhibitors, as all lung changes were mild and readily reversible.

Location is everything when it comes to megakaryocyte function.

Megakaryocytes (MKs) give rise to platelets, which are blood cells that are essential to prevent hemorrhage. Although the majority of MKs localize to the bone marrow, there is a distinct population of lung-residing MKs (MKL). In this issue of the JCI, Pariser et al. examined gene expression patterns of MKs collected from murine and nonhuman primate bone marrow or lung. This Commentary explores the premise that environmental factors from the lung determine the genetic and phenotypic similarity of MKL to lung dendritic cells, distinguishing MKL from bone marrow MKs. Indeed, while MKL retain the ability to make platelets, they also process and present antigens that activate CD4+ lymphocytes. These data suggest that MKL may play an important role in immune processes beyond platelet production.

More than just a platelet factory

Megakaryocytes are found in the lungs of mice and nonhuman primates, where they have a tissue-dependent antigen-presenting cell role and can activate CD4 + T cells.

An in vitro alveolar epithelial cell model recapitulates LRRK2 inhibitor-induced increases in lamellar body size observed in preclinical models

Alveolar type II (ATII) epithelial cells contain lamellar bodies (LBs) which synthesize and store lung surfactants. In animals, the inhibition or knockout of leucine-rich repeat kinase 2 (LRRK2) causes abnormal enlargement of LBs in ATII cells. This effect of LRRK2 inhibition in lung is largely accepted as being mediated directly through blocking of the kinase function; however, downstream consequences in the lung remain unknown. In this work we established an in vitro alveolar epithelial cell (AEC) model that recapitulates the in vivo phenotype of ATII cells and developed an assay to quantify changes in LB size in response to LRRK2 inhibitors. Culture of primary human AECs at the air-liquid interface on matrigel and collagen-coated transwell inserts in the presence of growth factors promoted the LB formation and apical microvilli and induced expression of LRRK2 and ATII cell markers. Treatment with a selective LRRK2 inhibitor resulted in pharmacological reduction of phospho-LRRK2 and a significant increase in LB size; effects previously reported in lungs of non-human primates treated with LRRK2 inhibitor. In summary, our human in vitro AEC model recapitulates the abnormal lung findings observed in LRRK2-perturbed animals and holds the potential for expanding current understanding of LRRK2 function in the lung.

LRRK2 inhibitors induce reversible changes in nonhuman primate lungs without measurable pulmonary deficits.

The kinase-activating mutation G2019S in leucine-rich repeat kinase 2 (LRRK2) is one of the most common genetic causes of Parkinson's disease (PD) and has spurred development of LRRK2 inhibitors. Preclinical studies have raised concerns about the safety of LRRK2 inhibitors due to histopathological changes in the lungs of nonhuman primates treated with two of these compounds. Here, we investigated whether these lung effects represented on-target pharmacology and whether they were reversible after drug withdrawal in macaques. We also examined whether treatment was associated with pulmonary function deficits. We conducted a 2-week repeat-dose toxicology study in macaques comparing three different LRRK2 inhibitors: GNE-7915 (30 mg/kg, twice daily as a positive control), MLi-2 (15 and 50 mg/kg, once daily), and PFE-360 (3 and 6 mg/kg, once daily). Subsets of animals dosed with GNE-7915 or MLi-2 were evaluated 2 weeks after drug withdrawal for lung function. All compounds induced mild cytoplasmic vacuolation of type II lung pneumocytes without signs of lung degeneration, implicating on-target pharmacology. At low doses of PFE-360 or MLi-2, there was ~50 or 100% LRRK2 inhibition in brain tissue, respectively, but histopathological lung changes were either absent or minimal. The lung effect was reversible after dosing ceased. Lung function tests demonstrated that the histological changes in lung tissue induced by MLi-2 and GNE-7915 did not result in pulmonary deficits. Our results suggest that the observed lung effects in nonhuman primates in response to LRRK2 inhibitors should not preclude clinical testing of these compounds for PD.

Pulmonary Neuroendocrine Cells Secrete γ-Aminobutyric Acid to Induce Goblet Cell Hyperplasia in Primate Models.

Mucus overproduction is a major contributor to morbidity and mortality in asthma. Mucus overproduction is induced by orchestrated actions of multiple factors that include inflammatory cytokines and γ-aminobutyric acid (GABA). GABA is produced only by pulmonary neuroendocrine cells (PNECs) in the mouse lung. Recent studies in a neonatal mouse model of allergic inflammation have shown that PNECs play an essential role in mucus overproduction by GABA hypersecretion. Whether PNECs mediate dysregulated GABA signaling for mucus overproduction in asthma is unknown. In this study, we characterized the cellular source of GABA in the lungs of nonhuman primates and humans and assessed GABA secretion and signaling in primate disease models. We found that like in mice, PNECs were the major source of GABA in primate lungs. In addition, an infant nonhuman primate model of asthma exhibited an increase in GABA secretion. Furthermore, subjects with asthma had elevated levels of expression of a subset of GABA type α (GABAα) and type β (GABAβ) receptors in airway epithelium compared with those of healthy control subjects. Last, employing a normal human bronchial epithelial cell model of preinduced mucus overproduction, we showed pharmaceutical blockade of GABAα and GABAβ receptor signaling reversed the effect of IL-13 on MUC5AC gene expression and goblet cell proliferation. Together, our data demonstrate an evolutionarily conserved intraepithelial GABA signaling that, in concert with IL-13, plays an essential role in mucus overproduction. Our findings may offer new strategies to ameliorate mucus overproduction in patients with asthma by targeting PNEC secretion and GABA signaling.

Topical application of nebulized human IgG, IgA and IgAM in the lungs of rats and non-human primates

BackgroundRecurrent and persistent infections are known to affect airways of patients with Primary Immunodeficiency despite appropriate replacement immunoglobulin serum levels. Interestingly, patients with Chronic Obstructive Pulmonary Disease or with non-CF bronchiectasis also show similar susceptibility to such infections. This may be due to the limited availability of immunoglobulins from the systemic circulation in the conductive airways, resulting in local immunodeficiency. Topical application of nebulized plasma-derived immunoglobulins may represent a means to address this deficiency. In this study, we assessed the feasibility of nebulizing plasma-derived immunoglobulins and delivering them into the airways of rats and non-human primates.MethodsDistinct human plasma-derived immunoglobulin isotype preparations were nebulized with an investigational eFlow® nebulizer and analyzed in vitro or deposited into animals. Biochemical and immunohistological analysis of nebulized immunoglobulins were then performed. Lastly, efficacy of topically applied human plasma-derived immunoglobulins was assessed in an acute Streptococcus pneumoniae respiratory infection in mice.ResultsCharacteristics of the resulting aerosols were comparable between preparations, even when using solutions with elevated viscosity. Neither the structural integrity nor the biological function of nebulized immunoglobulins were compromised by the nebulization process. In animal studies, immunoglobulins levels were assessed in plasma, broncho-alveolar lavages (BAL) and on lung sections of rats and non-human primates in samples collected up to 72 h following application. Nebulized immunoglobulins were detectable over 48 h in the BAL samples and up to 72 h on lung sections. Immunoglobulins recovered from BAL fluid up to 24 h after inhalation remained structurally and functionally intact. Importantly, topical application of human plasma-derived immunoglobulin G into the airways of mice offered significant protection against acute pneumococcal pneumonia.ConclusionTaken together our data demonstrate the feasibility of topically applying plasma-derived immunoglobulins into the lungs using a nebulized liquid formulation. Moreover, topically administered human plasma-derived immunoglobulins prevented acute respiratory infection.

Tuberculosis boosts HIV-1 production by macrophages through IL-10/STAT3 dependent tunneling nanotube formation

Abstract The tuberculosis (TB) bacillus, Mycobacterium tuberculosis (Mtb) and HIV-1 are known to act synergistically, however, the mechanisms by which Mtb exacerbates HIV-1 pathogenesis are not well known. Using in vitro and ex vivo cell culture system, we show that human M(IL-10) anti-inflammatory macrophages, present in TB-associated microenvironments, produced high levels of HIV-1. In vivo, M(IL-10) macrophages were expanded in lungs of co-infected non-human primates, their number correlated with disease severity, and markers for these cells (soluble CD163 and MerTK) accumulated in the blood of co-infected patients. These M(IL-10) macrophages formed direct cell-to-cell bridges, which we identified as tunneling nanotubes (TNTs) involved in viral transfer. TNT formation required the IL-10/STAT3 signaling pathway, and targeted inhibition of TNTs substantially reduced the enhanced HIV-1 cell-to-cell transfer and overproduction in M(IL-10) macrophages. Our study reveals that TNTs facilitate viral transfer and amplification, promoting TNT formation as a mechanism to be explored in TB/AIDS potential therapeutics.

Tuberculosis Exacerbates HIV-1 Infection through IL-10/STAT3-Dependent Tunneling Nanotube Formation in Macrophages

The tuberculosis (TB) bacillus, Mycobacterium tuberculosis (Mtb), and HIV-1 act synergistically; however, the mechanisms by which Mtb exacerbates HIV-1 pathogenesis are not well known. Usinginvitro and exvivo cell culture systems, weshow that human M(IL-10) anti-inflammatory macrophages, present in TB-associated microenvironment, produce high levels of HIV-1. Invivo,M(IL-10) macrophages are expanded in lungs of co-infected non-human primates, which correlates with disease severity. Furthermore, HIV-1/Mtb co-infected patients display an accumulation of M(IL-10) macrophage markers (soluble CD163 and MerTK). These M(IL-10) macrophages form direct cell-to-cell bridges, which we identifiedas tunneling nanotubes (TNTs) involved in viraltransfer. TNT formation requires the IL-10/STAT3 signaling pathway, and targeted inhibition of TNTssubstantially reduces the enhancement of HIV-1 cell-to-cell transfer and overproduction in M(IL-10) macrophages. Our study reveals that TNTs facilitate viral transfer and amplification, thereby promoting TNT formation as a mechanism to be explored in TB/AIDS potential therapeutics.