Optimization of Aspergillus versicolor Culture and Aerosolization in a Murine Model of Inhalational Fungal Exposure.

Aspergillus versicolor is ubiquitous in the environment and is particularly abundant in damp indoor spaces. Exposure to Aspergillus species, as well as other environmental fungi, has been linked to respiratory health outcomes, including asthma, allergy, and even local or disseminated infection. However, the pulmonary immunological mechanisms associated with repeated exposure to A. versicolor have remained relatively uncharacterized. Here, A. versicolor was cultured and desiccated on rice then placed in an acoustical generator system to achieve aerosolization. Mice were challenged with titrated doses of aerosolized conidia to examine deposition, lymphoproliferative properties, and immunotoxicological response to repeated inhalation exposures. The necessary dose to induce lymphoproliferation was identified, but not infection-like pathology. Further, it was determined that the dose was able to initiate localized immune responses. The data presented in this study demonstrate an optimized and reproducible method for delivering A. versicolor conidia to rodents via nose-only inhalation. Additionally, the feasibility of a long-term repeated exposure study was established. This experimental protocol can be used in future studies to investigate the physiological effects of repeated pulmonary exposure to fungal conidia utilizing a practical and relevant mode of delivery. In total, these data constitute an important foundation for subsequent research in the field.

CX3CR1-Expressing Myeloid Cells Regulate Host-Helminth Interaction and Lung Inflammation.

Many helminth life cycles, including hookworm, involve a mandatory lung phase, where myeloid and granulocyte subsets interact with the helminth and respond to infection-induced lung injury. To evaluate these innate subsets in Nippostrongylus brasiliensis infection, reporter mice for myeloid cells (CX3CR1GFP ) and granulocytes (PGRPdsRED ) are employed. Nippostrongylus infection induces lung infiltration of reporter cells, including CX3CR1+ myeloid cells and PGRP+ eosinophils. Strikingly, CX3CR1GFP/GFP mice, which are deficient in CX3CR1, are protected from Nippostrongylus infection with reduced weight loss, lung leukocyte infiltration, and worm burden compared to CX3CR1+/+ mice. This protective effect is specific for CX3CR1 as CCR2-deficient mice do not exhibit reduced worm burdens. Nippostrongylus co-culture with lung Ly6C+ monocytes or CD11c+ cells demonstrates that CX3CR1GFP/GFP monocytes secrete more pro-inflammatory cytokines and actively bind the parasites causing reduced motility. RNA sequencing of Ly6C+ or CD11c+ cells shows Nippostrongylus-induced gene expression changes, particularly in monocytes, associated with inflammation, chemotaxis, and extracellular matrix remodeling pathways. Analysis reveals cytotoxic and adhesion molecules as potential effectors against the parasite, such as Gzma and Gzmb, which are elevated in CX3CR1GFP/GFP monocytes. These studies validate a dual innate cell reporter for lung helminth infection and demonstrate that CX3CR1 impairs monocyte-helminth interaction.

Aspergillus versicolor Inhalation Triggers Neuroimmune, Glial, and Neuropeptide Transcriptional Changes.

Increasing evidence associates indoor fungal exposure with deleterious central nervous system (CNS) health, such as cognitive and emotional deficits in children and adults, but the specific mechanisms by which it might impact the brain are poorly understood. Mice were exposed to filtered air, heat-inactivated Aspergillus versicolor (3 × 105 spores), or viable A. versicolor (3 × 105 spores) via nose-only inhalation exposure 2 times per week for 1, 2, or 4 weeks. Analysis of cortex, midbrain, olfactory bulb, and cerebellum tissue from mice exposed to viable A. versicolor spores for 1, 2, and 4 weeks revealed significantly elevated pro-inflammatory (Tnf and Il1b) and glial activity (Gdnf and Cxc3r1) gene expression in several brain regions when compared to filtered air control, with the most consistent and pronounced neuroimmune response 48H following the 4-week exposure in the midbrain and frontal lobe. Bulk RNA-seq analysis of the midbrain tissue confirmed that 4 weeks of A. versicolor exposure resulted in significant transcriptional enrichment of several biological pathways compared to the filtered air control, including neuroinflammation, glial cell activation, and regulation of postsynaptic organization. Upregulation of Drd1, Penk, and Pdyn mRNA expression was confirmed in the 4-week A. versicolor exposed midbrain tissue, highlighting that gene expression important for neurotransmission was affected by repeated A. versicolor inhalation exposure. Taken together, these findings indicate that the brain can detect and respond to A. versicolor inhalation exposure with changes in neuroimmune and neurotransmission gene expression, providing much needed insight into how inhaled fungal exposures can affect CNS responses and regulate neuroimmune homeostasis.

Resolution of Pulmonary Inflammation Induced by Carbon Nanotubes and Fullerenes in Mice: Role of Macrophage Polarization.

Pulmonary exposure to certain engineered nanomaterials (ENMs) causes chronic lesions like fibrosis and cancer in animal models as a result of unresolved inflammation. Resolution of inflammation involves the time-dependent biosynthesis of lipid mediators (LMs)-in particular, specialized pro-resolving mediators (SPMs). To understand how ENM-induced pulmonary inflammation is resolved, we analyzed the inflammatory and pro-resolving responses to fibrogenic multi-walled carbon nanotubes (MWCNTs, Mitsui-7) and low-toxicity fullerenes (fullerene C60, C60F). Pharyngeal aspiration of MWCNTs at 40 µg/mouse or C60F at a dose above 640 µg/mouse elicited pulmonary effects in B6C3F1 mice. Both ENMs stimulated acute inflammation, predominated by neutrophils, in the lung at day 1, which transitioned to histiocytic inflammation by day 7. By day 28, the lesion in MWCNT-exposed mice progressed to fibrotic granulomas, whereas it remained as alveolar histiocytosis in C60F-exposed mice. Flow cytometric profiling of whole lung lavage (WLL) cells revealed that neutrophil recruitment was the greatest at day 1 and declined to 36.6% of that level in MWCNT- and 16.8% in C60F-treated mice by day 7, and to basal levels by day 28, suggesting a rapid initiation phase and an extended resolution phase. Both ENMs induced high levels of proinflammatory leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) with peaks at day 1, and high levels of SPMs resolvin D1 (RvD1) and E1 (RvE1) with peaks at day 7. MWCNTs and C60F induced time-dependent polarization of M1 macrophages with a peak at day 1 and subsequently of M2 macrophages with a peak at day 7 in the lung, accompanied by elevated levels of type 1 or type 2 cytokines, respectively. M1 macrophages exhibited preferential induction of arachidonate 5-lipoxygenase activating protein (ALOX5AP), whereas M2 macrophages had a high level expression of arachidonate 15-lipoxygenase (ALOX15). Polarization of macrophages in vitro differentially induced ALOX5AP in M1 macrophages or ALOX15 in M2 macrophages resulting in increased preferential biosynthesis of proinflammatory LMs or SPMs. MWCNTs increased the M1- or M2-specific production of LMs accordingly. These findings support a mechanism by which persistent ENM-induced neutrophilic inflammation is actively resolved through time-dependent polarization of macrophages and enhanced biosynthesis of specialized LMs via distinct ALOX pathways.

Morphology and quantification of fungal growth in residential dust and carpets.

Mold growth indoors is associated with negative human health effects, and this growth is limited by moisture availability. Dust deposited in carpet is an important source of human exposure due to potential elevated resuspension compared to hard floors. However, we need an improved understanding of fungal growth in dust and carpet to better estimate human exposure. The goal of this study was to compare fungal growth quantity and morphology in residential carpet under different environmental conditions, including equilibrium relative humidity (ERH) (50%, 85%, 90%, 95%, 100%), carpet fiber material (nylon, olefin, wool) and presence/absence of dust. We analyzed incubated carpet and dust samples from three Ohio homes for total fungal DNA, fungal allergen Alt a 1, and fungal morphology. Dust presence and elevated ERH (≥85%) were the most important variables that increased fungal growth. Elevated ERH increased mean fungal DNA concentration (P < 0.0001), for instance by approximately 1000 times at 100% compared to 50% ERH after two weeks. Microscopy also revealed more fungal growth at higher ERH. Fungal concentrations were up to 100 times higher in samples containing house dust compared to no dust. For fiber type, olefin had the least total fungal growth, and nylon had the most total fungi and A. alternata growth in unaltered dust. Increased ERH conditions were associated with increased Alt a 1 allergen concentration. The results of this study demonstrate that ERH, presence/absence of house dust, and carpet fiber type influence fungal growth and allergen production in residential carpet, which has implications for human exposure.

Inhalation of Stachybotrys chartarum Fragments Induces Pulmonary Arterial Remodeling.

Stachybotrys chartarum is a fungal contaminant within the built environment and a respiratory health concern in the United States. The objective of this study was to characterize the mechanisms influencing pulmonary immune responses to repeatedly inhaled S. chartarum. Groups of B6C3F1/N mice repeatedly inhaled viable trichothecene-producing S. chartarum conidia (strain A or strain B), heat-inactivated conidia, or high-efficiency particulate absolute-filtered air twice per week for 4 and 13 weeks. Strain A was found to produce higher amounts of respirable fragments than strain B. Lung tissue, serum, and BAL fluid were collected at 24 and 48 hours after final exposure and processed for histology, flow cytometry, and RNA and proteomic analyses. At 4 weeks after exposure, a T-helper cell type 2-mediated response was observed. After 13 weeks, a mixed T-cell response was observed after exposure to strain A compared with a T-helper cell type 2-mediated response after strain B exposure. After exposure, both strains induced pulmonary arterial remodeling at 13 weeks; however, strain A-exposed mice progressed more quickly than strain B-exposed mice. BAL fluid was composed primarily of eosinophils, neutrophils, and macrophages. Both the immune response and the observed pulmonary arterial remodeling were supported by specific cellular, molecular, and proteomic profiles. The immunopathological responses occurred earlier in mice exposed to high fragment-producing strain A. The rather striking induction of pulmonary remodeling by S. chartarum appears to be related to the presence of fungal fragments during exposure.

Cultivation and aerosolization of Stachybotrys chartarum for modeling pulmonary inhalation exposure.

Objective:Stachybotrys chartarum is a hydrophilic fungal species commonly found as a contaminant in water-damaged building materials. Although several studies have suggested that S. chartarum exposure elicits a variety of adverse health effects, the ability to characterize the pulmonary immune responses to exposure is limited by delivery methods that do not replicate environmental exposure. This study aimed to develop a method of S. chartarum aerosolization to better model inhalation exposures. Materials and methods: An acoustical generator system (AGS) was previously developed and utilized to aerosolize and deliver fungal spores to mice housed in a multi-animal nose-only exposure chamber. In this study, methods for cultivating, heat-inactivating, and aerosolizing two macrocyclic trichothecene-producing strains of S. chartartum using the AGS are described. Results and discussion: In addition to conidia, acoustical generation of one strain of S. chartarum resulted in the aerosolization of fungal fragments (<2 µm aerodynamic diameter) derived from conidia, phialides, and hyphae that initially comprised 50% of the total fungal particle count but was reduced to less than 10% over the duration of aerosolization. Acoustical generation of heat-inactivated S. chartarum did not result in a similar level of fragmentation. Delivery of dry, unextracted S. chartarum using these aerosolization methods resulted in pulmonary inflammation and immune cell infiltration in mice inhaling viable, but not heat-inactivated S. chartarum. Conclusions: These methods of S. chartarum growth and aerosolization allow for the delivery of fungal bioaerosols to rodents that may better simulate natural exposure within water-damaged indoor environments.

Non-traditional cytokines: How catecholamines and adipokines influence macrophages in immunity, metabolism and the central nervous system.

Catecholamines and adipokines function as hormones; catecholamines as neurotransmitters in the sympathetic nervous system, and adipokines as mediators of metabolic processes. It has become increasingly clear, however, that both also function as immunomodulators of innate and adaptive immune cells, including macrophages. Macrophages can respond to, as well as produce their own catecholamines. Dopamine, noradrenaline, and adrenaline are the most abundant catecholamines in the body, and can induce both pro-inflammatory and anti-inflammatory immune responses in macrophages, as well as non-immune processes such as thermogenesis. Though they are responsive to adipokines, particularly lipoproteins, leptin, and adiponectin, macrophages generally do not synthesize their own adipokines, with the exception being resistin-like molecules. Adipokines contribute to adverse metabolic and immune responses by stimulating lipid accumulation, foam cell formation and pro-inflammatory cytokine production in macrophages. Adipokines can also promote balance or resolution during metabolic and immune processes by promoting reverse lipid transport and expression of Th2 cytokines. This review will explore the mechanisms by which catecholamines and adipokines influence macrophage function in neural pathways, immunity and metabolism.

Macrophage-derived human resistin is induced in multiple helminth infections and promotes inflammatory monocytes and increased parasite burden.

Parasitic helminth infections can be associated with lifelong morbidity such as immune-mediated organ failure. A better understanding of the host immune response to helminths could provide new avenues to promote parasite clearance and/or alleviate infection-associated morbidity. Murine resistin-like molecules (RELM) exhibit pleiotropic functions following helminth infection including modulating the host immune response; however, the relevance of human RELM proteins in helminth infection is unknown. To examine the function of human resistin (hResistin), we utilized transgenic mice expressing the human resistin gene (hRetnTg+). Following infection with the helminth Nippostrongylus brasiliensis (Nb), hResistin expression was significantly upregulated in infected tissue. Compared to control hRetnTg- mice, hRetnTg+ mice suffered from exacerbated Nb-induced inflammation characterized by weight loss and increased infiltration of inflammatory monocytes in the lung, along with elevated Nb egg burdens and delayed parasite expulsion. Genome-wide transcriptional profiling of the infected tissue revealed that hResistin promoted expression of proinflammatory cytokines and genes downstream of toll-like receptor signaling. Moreover, hResistin preferentially bound lung monocytes, and exogenous treatment of mice with recombinant hResistin promoted monocyte recruitment and proinflammatory cytokine expression. In human studies, increased serum resistin was associated with higher parasite load in individuals infected with soil-transmitted helminths or filarial nematode Wuchereria bancrofti, and was positively correlated with proinflammatory cytokines. Together, these studies identify human resistin as a detrimental factor induced by multiple helminth infections, where it promotes proinflammatory cytokines and impedes parasite clearance. Targeting the resistin/proinflammatory cytokine immune axis may provide new diagnostic or treatment strategies for helminth infection and associated immune-mediated pathology.

Macrophage migration inhibitory factor is required for recruitment of scar-associated macrophages during liver fibrosis.

Recruitment of peripheral monocytes to the liver is a key contributor to the response to injury. MIF can act as a chemokine and cytokine, regulating innate immune responses in many tissues and cell types. We hypothesized that MIF contributes to the progression of CCl4-induced hepatic fibrosis by regulating recruitment of SAM. SAMs dynamically regulate HSC activation and ECM degradation. To gain insight into the role of MIF in progression of liver fibrosis, we investigated markers of fibrosis and immune responses after chronic CCl4 administration to female C57BL/6 and MIF(-/-) mice. Chronic CCl4 exposure increased activation of HSC in WT mice, indicated by increased expression of αSMA mRNA and protein, as well as mRNA for collagen 1α1; these responses were blunted in female MIF(-/-) mice. Despite lower activation of HSC in MIF(-/-) mice, accumulation of ECM was similar in WT and MIF(-/-)mice, suggesting a decreased rate of ECM degradation. Recruitment of SAMs was lower in MIF(-/-) mice compared with WT mice, both in their initial inflammatory phenotype, as well as in the later phase as proresolution macrophages. The decreased presence of resolution macrophages was associated with lower expression of MMP13 in MIF(-/-) mice. Taken together, these data indicate that MIF-dependent recruitment of SAMs contributes to degradation of ECM via MMP13, highlighting the importance of appropriate recruitment and phenotypic profile of macrophages in the resolution of fibrosis.

Innate immunity and cell death in alcoholic liver disease: role of cytochrome P4502E1.

Ethanol-induced liver injury is a complex process dependent upon the interaction of multiple cell types in the liver, as well as activation of the innate immune response. Increased expression of CYP2E1 in response to high concentrations of ethanol leads to greater production of cytotoxic ethanol metabolites, which in turn contribute to production of reactive oxygen species, oxidative stress, and ultimately, cell death. Necroptotic hepatocyte cell death in response to ethanol is mediated via a CYP2E1-dependent expression of receptor-interacting protein kinase 3 (RIP3), a key component of the necroptosome. In response to alarmins released during ethanol-induced necroptosis, the innate immune response is activated. Macrophage migration inhibitory factor (MIF), a pro-inflammatory multikine involved in many disease processes, is an essential component to this response to injury. MIF expression is increased during ethanol exposure via a CYP2E1-dependent pathway, likely contributing to an exacerbated innate immune response and chronic inflammation after chronic ethanol. This review will discuss the complex interactions between CYP2E1-dependent expression of RIP3 and MIF in the pathophysiology of chronic ethanol-induced liver injury.

Anaphylatoxin C5a modulates hepatic stellate cell migration.

BACKGROUND: C5a and its cognate receptor, C5a receptor (C5aR), key elements of complement, are critical modulators of liver immunity and fibrosis. However, the molecular mechanism for the cross talk between complement and liver fibrosis is not well understood. C5a is a potent chemokine regulating migration of cells in the innate immune system. Since activation and migration of hepatic stellate cells (HSC) are hallmarks of liver fibrosis, we hypothesized that C5a contributes to fibrosis by regulating HSC activation and/or migration. RESULTS: Primary cultures of mouse HSC increased expression of alpha smooth muscle actin (α-SMA) and collagen 1A (Col1A1) mRNA in response to activation on plastic. Expression of mRNA for C5aR, but not C5L2, a second C5a receptor that acts as a negative regulator, increased in parallel with markers of HSC activation in culture. Increased expression of C5aR on activated HSC was confirmed by immunocytochemistry. Cell surface expression of C5aR was also detected by flow cytometry on activated HSC isolated from mice expressing GFP under the control of the collagen promoter after exposure to chronic carbon tetrachloride. To understand the functional significance of C5aR expression in HSC, we next investigated whether C5a influenced HSC activation and/or migration. Challenge of HSC with C5a during culture had no effect on expression of α-SMA and Col1A1, suggesting that C5a did not influence HSC activation. Another important characteristic of HSC is their migratory capacity; migration of HSC in response to platelet derived growth factor (PDGF) and monocyte chemoattractant protein-1 (MCP-1) has been well characterized. Challenge of HSC with C5a enhanced HSC migration almost as efficiently as PDGF in a two-dimensional wound healing and Boyden chamber migration assays. C5a also stimulated expression of MCP-1. C5a-induced cell migration was slowed, but not completely inhibited, in presence of 227016, a MCP-1 receptor antagonist, suggesting C5a-induced migration occurs via both MCP-1-dependent and -independent mechanisms. CONCLUSIONS: These data reveal that C5a regulates migration of HSC and suggest a novel mechanism by which complement contributes to hepatic fibrosis. C5a and its receptors are therefore potential therapeutic targets for the prevention and/or treatment of liver fibrosis.

Macrophage migration inhibitory factor contributes to ethanol-induced liver injury by mediating cell injury, steatohepatitis, and steatosis.

UNLABELLED: Macrophage migration inhibitory factor (MIF), a multipotent protein that exhibits both cytokine and chemotactic properties, is expressed by many cell types, including hepatocytes and nonparenchymal cells. We hypothesized that MIF is a key contributor to liver injury after ethanol exposure. Female C57BL/6 or MIF-/- mice were fed an ethanol-containing liquid diet or pair-fed control diet for 4 (11% total kcal;early response) or 25 (32% kcal; chronic response) days. Expression of MIF messenger RNA (mRNA) was induced at both 4 days and 25 days of ethanol feeding. After chronic ethanol, hepatic triglycerides and plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were increased in wildtype, but not MIF-/-, mice. In order to understand the role of MIF in chronic ethanol-induced liver injury, we investigated the early response of wildtype and MIF-/- to ethanol. Ethanol feeding for 4 days increased apoptosis of hepatic macrophages and activated complement in both wildtype and MIF-/- mice. However, tumor necrosis factor alpha (TNF-α) expression was increased only in wildtype mice. This attenuation of TNF-α expression was associated with fewer F4/80+ macrophages in liver of MIF-/- mice. After 25 days of ethanol feeding, chemokine expression was increased in wildtype mice, but not MIF-/- mice. Again, this protection was associated with decreased F4/80+ cells in MIF-/- mice after ethanol feeding. Chronic ethanol feeding also sensitized wildtype, but not MIF-/-, mice to lipopolysaccharide, increasing chemokine expression and monocyte recruitment into the liver. CONCLUSION: Taken together, these data indicate that MIF is an important mediator in the regulation of chemokine production and immune cell infiltration in the liver during ethanol feeding and promotes ethanol-induced steatosis and hepatocyte damage.

Synergistic interaction between C5a and NOD2 signaling in the regulation of chemokine expression in RAW 264.7 macrophages.

The innate immune response is a complex process involving multiple pathogen-recognition receptors, including toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors. Complement is also a critical component of innate immunity. While complement is known to interact with TLR-mediated signals, the interactions between NOD-like receptors and complement are not well understood. Here we report a synergistic interaction between C5a and Nod2 signaling in RAW 264.7 macrophages. Long-term treatment with muramyl dipeptide (MDP), a NOD2 ligand, enhanced C5a-mediated expression of chemokine mRNAs in RAW 264.7 cells. This response was dependent on NOD2 expression and was associated with a decrease in expression of C5L2, a receptor for C5a which acts as a negative modulator of C5a receptor (C5aR) activity. MDP amplified C5a-mediated phosphorylation of p38 MAPK. Treatment of RAW264.7 cells with an inhibitor of p38 attenuated the synergistic effects of C5a on MDP-primed cells on MIP-2, but not MCP-1, mRNA. In contrast, inhibition of AKT prevented C5a stimulation of MCP-1, but not MIP-2, mRNA, in MDP-primed cells. Taken together, these data demonstrated a synergistic interaction between C5a and NOD2 in the regulation of chemokine expression in macrophages, associated with a down-regulation of C5L2, a negative regulator of C5a receptor activity.