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In epigenome-wide association studies analysing DNA methylation from samples containing multiple cell types, it is essential to adjust the analysis for cell type composition. One well established strategy for achieving this is reference-based cell type deconvolution, which relies on knowledge of the DNA methylation profiles of purified constituent cell types. These are then used to estimate the cell type proportions of each sample, which can then be incorporated to adjust the association analysis. Bronchoalveolar lavage is commonly used to sample the lung in clinical practice and contains a mixture of different cell types that can vary in proportion across samples, affecting the overall methylation profile. A current barrier to the use of bronchoalveolar lavage in DNA methylation-based research is the lack of reference DNA methylation profiles for each of the constituent cell types, thus making reference-based cell composition estimation difficult. Herein, we use bronchoalveolar lavage samples collected from children with cystic fibrosis to define DNA methylation profiles for the four most common and clinically relevant cell types: alveolar macrophages, granulocytes, lymphocytes and alveolar epithelial cells. We then demonstrate the use of these methylation profiles in conjunction with an established reference-based methylation deconvolution method to estimate the cell type composition of two different tissue types; a publicly available dataset derived from artificial blood-based cell mixtures and further bronchoalveolar lavage samples. The reference DNA methylation profiles developed in this work can be used for future reference-based cell type composition estimation of bronchoalveolar lavage. This will facilitate the use of this tissue in studies examining the role of DNA methylation in lung health and disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Shanthikumar, Neeland, Saffery, Ranganathan, Oshlack and Maksimovic.)

The occurrence of allergic diseases induced by aeroallergens has increased in the past decades. Among inhalant allergens, mites remain the important causal agent of allergic diseases. Storage mites- Tyrophagus putrescentiae are found in stored products or domestic environments. Major allergen Tyr-p3 plays a significant role in triggering IgE-mediated hypersensitivity. However, its effects on pulmonary inflammation, internalization, and activation in human epithelium remain elusive. Protease-activated receptors (PARs) are activated upon cleavage by proteases. A549 cells were used as an epithelial model to examine the PAR activation by Tyr-p3 and therapeutic potential of PAR-2 antagonist (GB88) in allergic responses. Enzymatic properties and allergen localization of Tyr-p3 were performed. The release of inflammatory mediators, phosphorylation of mitogen-activated protein kinase (MAPK), and cell junction disruptions were evaluated after Tyr-p3 challenge. Enzymatic properties determined by substrate digestion and protease inhibitors indicated that Tyr-p3 processes a trypsin-like serine protease activity. The PAR-2 mRNA levels were significantly increased by nTyr-p3 but inhibited by protease inhibitors or GB88. Protease allergen of nTyr-p3 significantly increased the levels of pro-inflammatory cytokines (IL-6 and TNF-α), chemokine (IL-8), and IL-1β in epithelial cells. nTyr-p3 markedly increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and MAP kinase. When cells were pretreated with GB88 then added nTyr-p3, the phosphorylated ERK1/2 did not inhibit by GB88. GB88 increased ERK1/2 phosphorylation in human epithelium cells. GB88 is able to block PAR-2-mediated calcium signaling which inhibits the nTyr-p3-induced Ca 2+ release. Among the pharmacologic inhibitors, the most effective inhibitor of the nTyr-p3 in the induction of IL-8 or IL-1β levels was GB88 followed by SBTI, MAPK/ERK, ERK, and p38 inhibitors. Levels of inflammatory mediators, including GM-CSF, VEGF, COX-2, TSLP, and IL-33 were reduced by treatment of GB88 or SBTI. Further, GB88 treatment down-regulated the nTyr-p3-induced PAR-2 expression in allergic patients with asthma or rhinitis. Tight junction and adherens junction were disrupted in epithelial cells by nTyr-p3 exposure; however, this effect was avoided by GB88. Immunostaining with frozen sections of the mite body showed the presence of Tyr-p3 throughout the intestinal digestive system, especially in the hindgut around the excretion site. In conclusion, our findings suggest that Tyr-p3 from domestic mites leads to disruption of the airway epithelial barrier after inhalation. Proteolytic activity of Tyr-p3 causes the PAR-2 mRNA expression, thus leading to the release of numerous inflammatory mediators. Antagonism of PAR2 activity suggests GB88 as the therapeutic potential for anti-inflammation medicine, especially in allergy development triggered by protease allergens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wang, Yu, Tsai, Yu and Liao.)

The respiratory tract is considered the main port of entry of Mycobacterium leprae , the causative agent of leprosy. However, the great majority of individuals exposed to the leprosy bacillus will never manifest the disease due to their capacity to develop protective immunity. Besides acting as a physical barrier, airway epithelium cells are recognized as key players by initiating a local innate immune response that orchestrates subsequent adaptive immunity to control airborne infections. However, to date, studies exploring the interaction of M. leprae with the respiratory epithelium have been scarce. In this work, the capacity of  M. leprae  to immune activate human alveolar epithelial cells was investigated, demonstrating that M. leprae -infected A549 cells secrete significantly increased IL-8 that is dependent on NF-κB activation. M. leprae was also able to induce IL-8 production in human primary nasal epithelial cells. M. leprae -treated A549 cells also showed higher expression levels of human β-defensin-2 (hβD-2), MCP-1, MHC-II and the co-stimulatory molecule CD80. Furthermore, the TLR-9 antagonist inhibited both the secretion of IL-8 and NF-κB activation in response to M. leprae , indicating that bacterial DNA sensing by this Toll-like receptor constitutes an important innate immune pathway activated by the pathogen. Finally, evidence is presented suggesting that extracellular DNA molecules anchored to Hlp, a histone-like protein present on the M. leprae surface, constitute major TLR-9 ligands triggering this pathway. The ability of M. leprae to immune activate respiratory epithelial cells herein demonstrated may represent a very early event during infection that could possibly be essential to the generation of a protective response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dias, Silva, Silva, Linhares, Santos, Vivarini, Marques, Rosa, Lopes, Berrêdo-Pinho and Pessolani.)

Respiratory diseases adversely affect infants and are the focus of efforts to develop vaccinations and other modalities to prevent disease. The infant immune system differs from that of older children and adults in many ways that are as yet ill understood. We have used a C57BL/6 mouse model of infection with a laboratory- adapted strain of influenza (PR8) to delineate the importance of the cytokine IL-6 in the innate response to primary infection and in the development of protective immunity in adult mice. Herein, we used this same model in infant (14 days of age) mice to determine the effect of IL-6 deficiency. Infant wild type mice are more susceptible than older mice to infection, similar to the findings in humans. IL-6 is expressed in the lung in the early response to PR8 infection. While intramuscular immunization does not protect against lethal challenge, intranasal administration of heat inactivated virus is protective and correlates with expression of IL-6 in the lung, activation of lung CD8 cells, and development of an influenza-specific antibody response. In IL-6 deficient mice, this response is abrogated, and deficient mice are not protected against lethal challenge. These studies support the importance of the role of the tissue environment in infant immunity, and further suggest that IL-6 may be helpful in the generation of protective immune responses in infants., (Copyright © 2020 Bonney, Krebs, Kim, Prakash, Torrance, Haynes and Rincon.)

The main function of the lung is to perform gas exchange while maintaining lung homeostasis despite environmental pathogenic and non-pathogenic elements contained in inhaled air. Resident cells must keep lung homeostasis and eliminate pathogens by inducing protective immune response and silently remove innocuous particles. Which lung cell type is crucial for this function is still subject to debate, with reports favoring either alveolar macrophages (AMs) or lung epithelial cells (ECs) including airway and alveolar ECs. AMs are the main immune cells in the lung in steady-state and their function is mainly to dampen inflammatory responses. In addition, they phagocytose inhaled particles and apoptotic cells and can initiate and resolve inflammatory responses to pathogens. Although AMs release a plethora of mediators that modulate immune responses, ECs also play an essential role as they are more than just a physical barrier. They produce anti-microbial peptides and can secrete a variety of mediators that can modulate immune responses and AM functions. Furthermore, ECs can maintain AMs in a quiescent state by expressing anti-inflammatory membrane proteins such as CD200. Thus, AMs and ECs are both very important to maintain lung homeostasis and have to coordinate their action to protect the organism against infection. Thus, AMs and lung ECs communicate with each other using different mechanisms including mediators, membrane glycoproteins and their receptors, gap junction channels, and extracellular vesicles. This review will revisit characteristics and functions of AMs and lung ECs as well as different communication mechanisms these cells utilize to maintain lung immune balance and response to pathogens. A better understanding of the cross-talk between AMs and lung ECs may help develop new therapeutic strategies for lung pathogenesis., (Copyright © 2020 Bissonnette, Lauzon-Joset, Debley and Ziegler.)

The opportunistic fungal pathogen Aspergillus fumigatus can cause severe infections, particularly in immunocompromised individuals. Upon infection, A. fumigatus faces the powerful and directly acting immune defense of the human host. The mechanisms on how A. fumigatus evades innate immune attack and complement are still poorly understood. Here, we identify A. fumigatus enolase, AfEno1, which was also characterized as fungal allergen, as a surface ligand for human plasma complement regulators. AfEno1 binds factor H, factor-H-like protein 1 (FHL-1), C4b binding protein (C4BP), and plasminogen. Factor H attaches to AfEno1 via two regions, via short conserved repeats (SCRs) 6-7 and 19-20, and FHL-1 contacts AfEno1 via SCRs 6-7. Both regulators when bound to AfEno1 retain cofactor activity and assist in C3b inactivation. Similarly, the classical pathway regulator C4BP binds to AfEno1 and bound to AfEno1; C4BP assists in C4b inactivation. Plasminogen which binds to AfEno1 via lysine residues is accessible for the tissue-type plasminogen activator (tPA), and active plasmin cleaves the chromogenic substrate S2251, degrades fibrinogen, and inactivates C3 and C3b. Plasmin attached to swollen A. fumigatus conidia damages human A549 lung epithelial cells, reduces the cellular metabolic activity, and induces cell retraction, which results in exposure of the extracellular matrix. Thus, A. fumigatus AfEno1 is a moonlighting protein and virulence factor which recruits several human regulators. The attached human regulators allow the fungal pathogen to control complement at the level of C3 and to damage endothelial cell layers and tissue components., (Copyright © 2019 Dasari, Koleci, Shopova, Wartenberg, Beyersdorf, Dietrich, Sahagún-Ruiz, Figge, Skerka, Brakhage and Zipfel.)

Allergens are molecules that elicit a hypersensitive inflammatory response in sensitized individuals and are derived from a variety of sources. Alt a 1 is the most clinically important secreted allergen of the ubiquitous fungus, Alternaria . It has been shown to be a major allergen causing IgE-mediated allergic response in the vast majority of Alternaria -sensitized individuals. However, no studies have been conducted in regards to the innate immune eliciting activities of this clinically relevant protein. In this study, recombinant Alt a 1 was produced, purified, labeled, and incubated with BEAS-2B, NHBE, and DHBE human lung epithelial cells. Alt a 1 elicited strong induction of IL-8, MCP-1, and Gro-a/b/g. Using gene-specific siRNAs, blocking antibodies, and chemical inhibitors such as LPS-RS, it was determined that Alt a 1-induced immune responses were dependent upon toll-like receptors (TLRs) 2 and 4, and the adaptor proteins MYD88 and TIRAP. Studies utilizing human embryonic kidney cells engineered to express single receptors on the cell surface such as TLRs, further confirmed that Alt a 1-induced innate immunity is dependent upon TLR4 and to a lesser extent TLR2.

Inflammatory lung responses are one of the characterized features in the pathogenesis of many lung diseases, including acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD). Alveolar macrophages (AMs) and alveolar epithelial cells are the first line of host defense and innate immunity. Due to their central roles in both the initiation and resolution of inflammatory lung responses, AMs constantly communicate with other lung cells, including the alveolar epithelial cells. In the past, emerging evidence suggests that extracellular vesicles play an essential role in cell-cell crosstalk. In this review, we will discuss the recent findings on the intercellular communications between lung epithelial cells and alveolar macrophages, via EV-mediated signal transfer.