Citations

Abstract Fetal-derived tissue-resident macrophages exhibit stem cell-like features of self-renewal in adulthood to maintain macrophage population during homeostasis and/or various insults. However, little is known about the cellular and molecular mechanisms modulating proliferative and inflammatory fate decisions of tissue-resident macrophages in vivo. Here, we show that WNT-β-catenin signaling inhibited lung-resident alveolar macrophage (AM) self-renewal, while simultaneously promoted AM inflammatory activities in vitro and in vivo during influenza virus infection. Mechanistically, WNT engagement facilitated the binding of β-catenin with HIF-1α over its conventional binding partner TCF-4. Such a binding choice led to the elevated macrophage inflammation in a glycolysis-dependent manner, while inhibited AM self-renewal by causing mitochondrial damage and impairing oxidative phosphorylation. Thus, AM self-renewal and inflammatory activity are uncoupled by WNT-β-catenin signaling through HIF-1α-mediated cellular metabolic choice. Importantly, we showed that AM with high HIF-1α activity had limited proliferative capacity and produced inflammatory cytokines, while AM with low HIF-1α activity were highly proliferative and expressed genes associated with tissue repair function in vivo during influenza virus infection. In accordance, we demonstrated that AM proliferation and repopulation were needed for optimal lung repair following the clearance of influenza virus in the respiratory tract. Our results have revealed key mechanisms modulating macrophage fate choice between progeny production versus inflammatory effector activity, and subsequent effects on tissue inflammation and repair.

Abstract Alveolar macrophages (AMs) are the most abundant immune cells in the lung during homeostasis. AMs are critical for normal lung development, surfactant metabolism and host defense against various respiratory pathogens. Despite being terminally differentiated cells, AMs are able to proliferate and self-renew to maintain their compartment without the input of the hematopoietic system in the adulthood during homeostasis. However, the molecular and metabolic mechanisms modulating AM proliferative responses are still incompletely understood. Here we have investigated the metabolic regulation of AM proliferation and self-renewal. We found that mitochondrial metabolism is critical for AM proliferation in vitro and in vivo. Interestingly, inhibition of glucose uptake or fatty acid oxidation did not significantly impact AM proliferation. Rather, inhibition of the glutamine metabolic process abolished AM proliferation in vitro and in vivo following their partial depletion during influenza infection. We further demonstrated that glutamine metabolism was required for AM mitochondrial respiration. Our data indicate that glutamine is a critical substrate fueling mitochondrial metabolism required for AM proliferation. Our study is expected to shed light on the metabolic regulation of AM maintenance and repopulation during homeostasis and following acute respiratory viral infection.

Abstract Sialic acids are the terminal modification on glycoproteins and glycolipids. The sialic acid transferase ST8Sia6 generates a2,8-linked disialic acids on O-linked glycoproteins, which bind to the inhibitory receptor Siglec-E on innate immune cells and suppress immune activation. Recently, our lab has shown that ST8Sia6 expression on tumor cells inhibits the immune response through Siglec-E, leading to increased tumor growth and decreased survival. Therefore, the products of ST8Sia6 can modulate the immune response to tumors. Our lab has generated ST8Sia6 knockout (KO) mice, and we seek to understand the role of ST8Sia6 in regulating the immune response to pathogens. We find that ST8Sia6 KO mice, when challenged with influenza virus, clear the infection significantly faster than wildtype controls, suggesting that absence of ST8Sia6 reduces inhibition of the innate compartment, thereby increasing immune recruitment to infection. This data suggests an important role for interactions between Siglec-E and ST8Sia6-generated disialic acids in immune regulation and recruitment to infection sites.

Abstract Alveolar Macrophages (AMs) are unique innate immune cells that reside in the alveolar space and accommodate the ever-changing need of the lungs against internal and external challenges. During homeostasis, AMs maintained themselves through self-renewal without the need for input from adult hematopoietic stem cells. Currently, little is known about how aging influences AM dynamics, heterogeneity and self-renewal. To this end, we have performed single-cell RNA sequencing (scRNA-seq) on murine lung AMs (CD11+ Siglec F+) isolated from young (~8w) and aged (~2y) mice. We discovered that despite similar transcriptome in the proliferating cells, AMs from aged mice had reduced proliferation and self-renew ability than those from the young mice. AMs from aged mice also displayed elevated senescence markers such as p16, p21 and p53. Consistently, AMs from aged mice had reduced DNA repair ability, which could contribute to their impaired capacity to pass through the cell cycle checkpoints. Interestingly, AMs from aged mice also possessed reduced pro-apoptotic gene profile. Thus, we hypothesize that the reduced proliferation- and apoptosis-associated gene profiles in aged AMs may lead to the enhanced senescence-like phenotype in those cells. Further validation of the senescence-like phenotype of aged AMs and investigation of how they contribute to lung “inflammaging” are warranted.

Abstract Severe influenza A virus infection typically triggers excessive and detrimental lung inflammation with massive cell infiltration and hyper-production of cytokines and chemokines. Defining research models to alleviate lung damage without compromising viral control is critical. We identified a novel function for nuclear matrix protein 4 (NMP4), a zinc-finger-containing transcription factor in regulating lung inflammation and antiviral innate immune responses. Nmp4-deficient mice are protected from H1N1 influenza infection, losing less body weight compared to the wild-type mice. Nmp4-deficiency does not affect viral clearance or adaptive immune responses, but significantly reduces the accumulation of neutrophils and monocytes in the lungs and the early inflammation. Consistent with fewer innate cells in the airways, infected Nmp4-deficient mice show decreased expression of chemokine genes Ccl2 and Cxcl1 as well as pro-inflammatory cytokine genes Il1b and Il6. We further reveal that NMP4 binds to the promoters and/or conserved non-coding sequences (CNS) of Ccl2 and Cxcl1 in mouse lung epithelial cells. Besides, the knockdown of NMP4 by CRISPR/Cas9 or siRNA significantly lessens the luciferase activities of Ccl2 and Cxcl1 promoters and/or CNS. Taken together, our data suggest that NMP4 regulates monocyte- and neutrophil-attracting chemokine expression in airway epithelial cells upon influenza infection, resulting in exaggerated innate inflammation and lung tissue damage.

Abstract CD8+ tissue-resident memory T (TRM) cells provide frontline immunity in mucosal tissues. The mechanisms regulating CD8+ TRM maintenance, heterogeneity, and protective and pathological functions are largely elusive. Here, we identify a population of CD8+ TRM cells that is maintained by major histocompatibility complex class I (MHC-I) signaling, and CD80 and CD86 co-stimulation after acute influenza infection. These TRM cells have both “exhausted-like” phenotypes and memory features, and provide heterologous immunity against secondary infection. Using recombinant influenza virus, we demonstrate that the development of these PD-1hi “exhausted-like” TRM cells is dependent on the doses of the antigen during primary infection. Furthermore, we showed that lung CD11c+ antigen presenting cells are required for sustaining these TRM cells at the memory stage. Our data suggest that continuous antigen presentation in situ is required for the development and maintenance of these “exhausted-like” TRM cells in the lung. Strikingly, PD-L1 blockade after the resolution of primary infection promotes the rejuvenation of these “exhausted-like” TRM cells, restoring protective immunity at the cost of promoting post-infection inflammatory and fibrotic sequelae. Thus, PD-1 serves to limit the pathogenic capacity of “exhausted-like” TRM cells at the memory phase under specific tissue environment with chronic low-levels of antigen presentation following viral clearance. Our data indicate that TRM cell “exhaustion” is the result of a tissue-specific cellular adaptation that balances fibrotic sequelae with protective immunity.

Abstract Infants born prematurely often develop chronic lung diseases including bronchopulmonary dysplasia (BPD). BPD is associated with greatly increased host susceptibility and severe disease development following respiratory viral infection. To this end, we have recently established a neonatal hyperoxia model respiratory viral infection in hosts with lung prematurity and BPD. Using this model, we observed that mice exposed with neonatal hyperoxia (80% oxygen, BPD hosts) developed enhanced diseases following influenza virus or respiratory syncytial virus (RSV) infection compared to those of influenza or RSV-infected control mice. We showed that neonatal hyperoxia leaded to modestly enhanced viral replication in the respiratory tract. Using nanostring immune gene profiling and multiplex cytokine analysis, we found that neonatal hyperoxia markedly enhanced immune-associated gene expression and the production of a number of proinflammatory cytokines and chemokines in the airway compared to those of control mice. Neonatal hyperoxia also increased the accumulation of multiple inflammatory innate cell types that were shown to be pathogenic during respiratory viral infections. Importantly, antiviral T, B cells and NK cells were similar in the lungs between infected neonatal hyperoxia-exposed and control mice, which are consistent with the similar viral clearance kinetics in those mice. Thus, we have successfully established a clinically-relevant mouse model of respiratory viral infection in BPD hosts. We expect to further uncover novel insights on the viral pathogenesis and host disease devlopment in this novel model of respiratory viral infection in hosts with chronic lung diseases.

Abstract Tissue-resident memory CD8+ T cells (TRM) share characteristics and core residency gene programs with tumor-infiltrating lymphocytes (TILs). However, the transcriptional, metabolic and epigenetic regulation of TRM and TIL differentiation, maintenance and function are largely undefined. Furthermore, it is unknown whether metabolic and epigenetic manipulations can be employed to promote TRM/TIL functional programs for cancer immunotherapy. Here we report a critical mechanism programming the mitochondrial and epigenetic regulation of TRM/TIL functionality in situ. We find that mouse and human TRM/TIL express the transcription factor BHLHE40 and the BHLHE40-associated gene signature. Bhlhe40 is specifically required for TRM and TIL development and poly-functionality. Local PD-1 signaling inhibits TIL Bhlhe40 expression, and Bhlhe40 is critical for TIL reinvigoration following anti-PD therapy. Mechanistically, Bhlhe40 sustains TRM/TIL mitochondrial fitness for the promotion of a functional epigenetic state. Building on these findings, we also identify an epigenetic and metabolic regimen that promotes TRM/TIL gene signatures associated with tissue residency and poly-functionality through in vitro T cell screening. Strikingly, this regimen empowers the anti-tumor activity of CD8+ T cells and possesses therapeutic potential even at an advanced tumor stage in mouse models. Our results provide mechanistic insights on the local regulation of TRM and TIL function, and offer a viable strategy for developing novel immunotherapeutic means for cancer.

Abstract Alveolar Macrophages (AMs) are a unique population of innate immune cells that reside in the alveolar space and contribute to accommodate the ever-changing need of the lungs against the external environmental challenges. AMs derive from the fetal origins and are capable of maintaining themselves through self-renewal without the input of hematopoiesis during the adulthood. However, little is known about how aging is influencing the molecular characteristics and heterogeneity of the AMs during homeostasis. Using single-cell RNA sequencing (scRNA-seq) and Seurat clustering, we discovered AMs can be subdivided into different “subsets” with unique molecular characteristics during homeostasis. Interestingly, when we further investigate the AM population with the combined data from the young(~8w) and old(~2y) mice, the data suggested that the proliferating cell fall in the same cluster with reduced number in the old-mice group, while the poised inflammatory cells showed distinct molecular patterns between the group with different age. We hypothesize that the inflammatory and the self-renewal AM subpopulations may be maintained by distinct extracellular signals. With the progress of aging, the self-renewal ability of AMs would reduce because of the number of proliferating cells but not the change of molecular patterns. At the same time, the inflammatory population would change its characteristics with age, and potentially lead to functional change upon activation. Further validation of the AM subset identity as well as the developmental and functional interrelationship of these subsets are required. In addition, the extrinsic and intrinsic signals responsible for the maintenance of these AM subsets are to be explored.

Abstract The role of Bcl6 expression in different immune compartments in regulating anti-tumor immunity is relatively unknown. Previous studies have linked Bcl6 expression in PBMC with poor prognosis in non-small cell lung cancer. We found that high expression of Bcl6 in tumor predicts survival in gastric and ovarian cancers. Using cell type specific conditional knockouts, we were able to investigate the contribution of Bcl6 expression in different lineages in modulating immune response to subcutaneously implanted B16-ova melanoma tumors. Myeloid deficiency of Bcl6 results in decreased tumor growth and metastasis. This is accompanied by enrichment of antigen specific T cells within the tumor. These tumor infiltrating T cells show an increased expression of exhausted markers such as PD-1, Lag-3 and Tim-3. Tumor associated innate cells also upregulate PD-L1. Furthermore, the TILs display an enhanced effector profile measured by robust production of granzyme B, interferon gamma and CCL3 on ex vivo stimulation. This is accompanied by increase in poly-functional CD8 T cells. Our result suggests that Bcl6 expression in the monocyte/macrophage lineage dampens the development of effective anti-tumor immunity and augments immunosuppressive tumor microenvironment. We will elucidate the exact underlying mechanisms and evaluate the translational potential of Bcl6 inhibitors in conjunction with immune checkpoint blockade in the future.