The circadian system is an important regulator of immune function. Human inflammatory lung diseases frequently show time-of-day variation in symptom severity and lung function, but the mechanisms and ...cell types underlying these effects remain unclear. We show that pulmonary antibacterial responses are modulated by a circadian clock within epithelial club (Clara) cells. These drive circadian neutrophil recruitment to the lung via the chemokine CXCL5. Genetic ablation of the clock gene Bmal1 (also called Arntl or MOP3) in bronchiolar cells disrupts rhythmic Cxcl5 expression, resulting in exaggerated inflammatory responses to lipopolysaccharide and an impaired host response to Streptococcus pneumoniae infection. Adrenalectomy blocks rhythmic inflammatory responses and the circadian regulation of CXCL5, suggesting a key role for the adrenal axis in driving CXCL5 expression and pulmonary neutrophil recruitment. Glucocorticoid receptor occupancy at the Cxcl5 locus shows circadian oscillations, but this is disrupted in mice with bronchiole-specific ablation of Bmal1, leading to enhanced CXCL5 expression despite normal corticosteroid secretion. The therapeutic effects of the synthetic glucocorticoid dexamethasone depend on intact clock function in the airway. We now define a regulatory mechanism that links the circadian clock and glucocorticoid hormones to control both time-of-day variation and the magnitude of pulmonary inflammation and responses to bacterial infection.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The role of myeloid cells in supporting cancer growth is well established. Most work has focused on myeloid-derived suppressor cells (MDSC) that accumulate in tumor-bearing animals, but ...tumor-associated neutrophils (TAN) are also known to be capable of augmenting tumor growth. However, little is known about their evolution, phenotype, and relationship to naïve neutrophils (NN) and to the granulocytic fraction of MDSC (G-MDSC).In the current study, a transcriptomics approach was used in mice to compare these cell types. Our data show that the three populations of neutrophils are significantly different in their mRNA profiles with NN and G-MDSC being more closely related to each other than to TAN. Structural genes and genes related to cell-cytotoxicity (i.e. respiratory burst) were significantly down-regulated in TAN. In contrast, many immune-related genes and pathways, including genes related to the antigen presenting complex (e.g. all six MHC-II complex genes), and cytokines (e.g. TNF-α, IL-1-α/β), were up-regulated in G-MDSC, and further up-regulated in TAN. Thirteen of the 25 chemokines tested were markedly up-regulated in TAN compared to NN, including striking up-regulation of chemoattractants for T/B-cells, neutrophils and macrophages.This study characterizes different populations of neutrophils related to cancer, pointing out the major differences between TAN and the other neutrophil populations.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Neonatal colonization by microbes, which begins immediately after birth, is influenced by gestational age and the mother's microbiota and is modified by exposure to antibiotics. In neonates, ...prolonged duration of antibiotic therapy is associated with increased risk of late-onset sepsis (LOS), a disorder controlled by neutrophils. A role for the microbiota in regulating neutrophil development and susceptibility to sepsis in the neonate remains unclear. We exposed pregnant mouse dams to antibiotics in drinking water to limit transfer of maternal microbes to the neonates. Antibiotic exposure of dams decreased the total number and composition of microbes in the intestine of the neonates. This was associated with decreased numbers of circulating and bone marrow neutrophils and granulocyte/macrophage-restricted progenitor cells in the bone marrow of antibiotic-treated and germ-free neonates. Antibiotic exposure of dams reduced the number of interleukin-17 (IL-17)-producing cells in the intestine and production of granulocyte colony-stimulating factor (G-CSF). Granulocytopenia was associated with impaired host defense and increased susceptibility to Escherichia coli K1 and Klebsiella pneumoniae sepsis in antibiotic-treated neonates, which could be partially reversed by administration of G-CSF. Transfer of a normal microbiota into antibiotic-treated neonates induced IL-17 production by group 3 innate lymphoid cells (ILCs) in the intestine, increasing plasma G-CSF levels and neutrophil numbers in a Toll-like receptor 4 (TLR4)- and myeloid differentiation factor 88 (MyD88)-dependent manner and restored IL-17-dependent resistance to sepsis. Specific depletion of ILCs prevented IL-17- and G-CSF-dependent granulocytosis and resistance to sepsis. These data support a role for the intestinal microbiota in regulation of granulocytosis, neutrophil homeostasis and host resistance to sepsis in neonates.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Red blood cells (RBCs) are essential for aerobic respiration through delivery of oxygen to distant tissues. However, RBCs are currently considered immunologically inert, and few, if any, secondary ...functions of RBCs have been identified. Here, we showed that RBCs serve as critical immune sensors through surface expression of the nucleic acid–sensing Toll-like receptor 9 (TLR9). Mammalian RBCs expressed TLR9 on their surface and bound CpG-containing DNA derived from bacteria, plasmodia, and mitochondria. RBC-bound mitochondrial DNA was increased during human and murine sepsis and pneumonia. In vivo, CpG-carrying RBCs drove accelerated erythrophagocytosis and innate immune activation characterized by increased interferon signaling. Erythroid-specific deletion of TLR9 abrogated erythrophagocytosis and decreased local and systemic cytokine production during CpG-induced inflammation and polymicrobial sepsis. Thus, detection and capture of nucleic acid by TLR9-expressing RBCs regulated red cell clearance and inflammatory cytokine production, demonstrating that RBCs function as immune sentinels during pathologic states. Consistent with these findings, RBC-bound mitochondrial DNA was elevated in individuals with viral pneumonia and sepsis secondary to coronavirus disease 2019 (COVID-19) and associated with anemia and severity of disease. These findings uncover a previously unappreciated role of RBCs as critical players in inflammation distinct from their function in gas transport.
TGF-β blockade significantly slows tumor growth through many mechanisms, including activation of CD8
+ T cells and macrophages. Here, we show that TGF-β blockade also increases neutrophil-attracting ...chemokines, resulting in an influx of CD11b
+/Ly6G
+ tumor-associated neutrophils (TANs) that are hypersegmented, more cytotoxic to tumor cells, and express higher levels of proinflammatory cytokines. Accordingly, following TGF-β blockade, depletion of these neutrophils significantly blunts antitumor effects of treatment and reduces CD8
+ T cell activation. In contrast, in control tumors, neutrophil depletion decreases tumor growth and results in more activated CD8
+ T cells intratumorally. Together, these data suggest that TGF-β within the tumor microenvironment induces a population of TAN with a protumor phenotype. TGF-β blockade results in the recruitment and activation of TANs with an antitumor phenotype.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Type II alveolar cells (AT2s) are critical for basic respiratory homeostasis and tissue repair after lung injury. Prior studies indicate that AT2s also express major histocompatibility complex ...class II (MHCII) molecules, but how MHCII expression by AT2s is regulated and how it contributes to host defense remain unclear. Here we show that AT2s express high levels of MHCII independent of conventional inflammatory stimuli, and that selective loss of MHCII from AT2s in mice results in modest worsening of respiratory virus disease following influenza and Sendai virus infections. We also find that AT2s exhibit MHCII presentation capacity that is substantially limited compared to professional antigen presenting cells. The combination of constitutive MHCII expression and restrained antigen presentation may position AT2s to contribute to lung adaptive immune responses in a measured fashion, without over-amplifying damaging inflammation.
Alveolar epithelial regeneration is essential for recovery from devastating lung diseases. This process occurs when type II alveolar pneumocytes (AT2 cells) proliferate and transdifferentiate into ...type I alveolar pneumocytes (AT1 cells). We used genome-wide analysis of chromatin accessibility and gene expression following acute lung injury to elucidate repair mechanisms. AT2 chromatin accessibility changed substantially following injury to reveal STAT3 binding motifs adjacent to genes that regulate essential regenerative pathways. Single-cell transcriptome analysis identified brain-derived neurotrophic factor (Bdnf) as a STAT3 target gene with newly accessible chromatin in a unique population of regenerating AT2 cells. Furthermore, the BDNF receptor tropomyosin receptor kinase B (TrkB) was enriched on mesenchymal alveolar niche cells (MANCs). Loss or blockade of AT2-specific Stat3, Bdnf or mesenchyme-specific TrkB compromised repair and reduced Fgf7 expression by niche cells. A TrkB agonist improved outcomes in vivo following lung injury. These data highlight the biological and therapeutic importance of the STAT3-BDNF-TrkB axis in orchestrating alveolar epithelial regeneration.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses. Small basic proteins compact and regulate both viral and cellular DNA genomes. ...Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.
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IJS, KISLJ, NUK, SBMB, UL, UM, UPUK
Neutrophil extracellular traps (NETs) provide host defense, but can contribute to the pathobiology of diverse human diseases. We sought to determine the extent and mechanism by which NETs contribute ...to human airway cell inflammation.
Primary normal human bronchial epithelia (HBEs) grown at air liquid interface and wtCFBE41o- cells (expressing wtCFTR) were exposed to cell-free NETs from unrelated healthy volunteers for 18 hours in vitro. Cytokines were measured in the apical supernatant by Luminex and the effect on the HBE transcriptome was assessed by RNA seq.
NETs consistently stimulated IL-8, TNF-α and IL-1α secretion by HBEs from multiple donors, with variable effects on other cytokines (IL-6, G-CSF, GM-CSF). HBE RNAs encoding IL-1 family cytokines, particularly IL-36 subfamily members, were increased in response to NETs. NET exposure in the presence of anakinra (recombinant human IL-1RA) dampened NET-induced changes in IL-8 and TNF-α proteins as well as IL-36α RNA. RhIL-36RA limited the increase in proinflammatory cytokine RNAs in HBEs exposed to NETs.
NETs selectively upregulate an IL-1 family cytokine response in HBEs, which enhances IL-8 production and is limited by rhIL-1RA. Present findings describe a unique mechanism by which NETs may contribute to inflammation in human lung disease in vivo. NET-driven IL-1 signaling may represent a novel target for modulating inflammation in diseases characterized by a substantial NET burden.
Potentially hazardous CpG-containing cell-free mitochondrial DNA (cf-mtDNA) is routinely released into the circulation and is associated with morbidity and mortality in critically ill patients. How ...the body avoids inappropriate innate immune activation by cf-mtDNA remains unknown. Because red blood cells (RBCs) modulate innate immune responses by scavenging chemokines, we hypothesized that RBCs may attenuate CpG-induced lung inflammation through direct scavenging of CpG-containing DNA.
To determine the mechanisms of CpG-DNA binding to RBCs and the effects of RBC-mediated DNA scavenging on lung inflammation.
mtDNA on murine RBCs was measured under basal conditions and after systemic inflammation. mtDNA content on human RBCs from healthy control subjects and trauma patients was measured. Toll-like receptor 9 (TLR9) expression on RBCs and TLR9-dependent binding of CpG-DNA to RBCs were determined. A murine model of RBC transfusion after CpG-DNA-induced lung injury was used to investigate the role of RBC-mediated DNA scavenging in mitigating lung injury in vivo.
Under basal conditions, RBCs bind CpG-DNA. The plasma-to-RBC mtDNA ratio is low in naive mice and in healthy volunteers but increases after systemic inflammation, demonstrating that the majority of cf-mtDNA is RBC-bound under homeostatic conditions and that the unbound fraction increases during inflammation. RBCs express TLR9 and bind CpG-DNA through TLR9. Loss of TLR9-dependent RBC-mediated CpG-DNA scavenging increased lung injury in vivo.
RBCs homeostatically bind mtDNA, and RBC-mediated DNA scavenging is essential in mitigating lung injury after CpG-DNA. Our data suggest a role for RBCs in regulating lung inflammation during disease states where cf-mtDNA is elevated, such as sepsis and trauma.
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