Review article: New developments in biomarkers and clinical drug development in alpha-1 antitrypsin deficiency-related liver disease.

BACKGROUND: Alpha-1 antitrypsin liver disease (AATLD) occurs in a subset of patients with alpha-1 antitrypsin deficiency. Risk factors for disease progression and specific pathophysiologic features are not well known and validated non-invasive assessments for disease severity are lacking. Currently, there are no approved treatments for AATLD. AIMS: To outline existing understanding of AATLD and to identify knowledge gaps critical to improving clinical trial design and development of new treatments. METHODS: This report was developed following a multi-stakeholder forum organised by the Alpha-1 Antitrypsin Deficiency Related Liver Disease Expert Panel in which experts presented an overview of the available literature on this topic. RESULTS: AATLD results from a 'gain of toxic function' and primarily manifests in those with the homozygous Pi*ZZ genotype. Accumulation of misfolded 'Z' AAT protein in liver cells triggers intracellular hepatocyte injury which may ultimately lead to hepatic fibrosis. Male gender, age over 50 years, persistently elevated liver tests, concomitant hepatitis B or C virus infection, and metabolic syndrome, including obesity and type 2 diabetes mellitus, are known risk factors for adult AATLD. While the gold standard for assessing AATLD disease activity is liver histology, less invasive measures with low intra- and inter-observer variability are needed. Measurement of liver stiffness shows promise; validated thresholds for staging AATLD are in development. Such advances will help patients by enabling risk stratification and personalised surveillance, along with streamlining the development process for novel therapies. CONCLUSIONS: This inaugural forum generated a list of recommendations to address unmet needs in the field of AATLD.

Lower Airway Dysbiosis Augments Lung Inflammatory Injury in Mild-to-Moderate Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is associated with high morbidity, mortality, and healthcare costs. Cigarette smoke is a causative factor; however, not all heavy smokers develop COPD. Microbial colonization and infections are contributing factors to disease progression in advanced stages. Objectives: We investigated whether lower airway dysbiosis occurs in mild-to-moderate COPD and analyzed possible mechanistic contributions to COPD pathogenesis. Methods: We recruited 57 patients with a >10 pack-year smoking history: 26 had physiological evidence of COPD, and 31 had normal lung function (smoker control subjects). Bronchoscopy sampled the upper airways, lower airways, and environmental background. Samples were analyzed by 16S rRNA gene sequencing, whole genome, RNA metatranscriptome, and host RNA transcriptome. A preclinical mouse model was used to evaluate the contributions of cigarette smoke and dysbiosis on lower airway inflammatory injury. Measurements and Main Results: Compared with smoker control subjects, microbiome analyses showed that the lower airways of subjects with COPD were enriched with common oral commensals. The lower airway host transcriptomics demonstrated differences in markers of inflammation and tumorigenesis, such as upregulation of IL-17, IL-6, ERK/MAPK, PI3K, MUC1, and MUC4 in mild-to-moderate COPD. Finally, in a preclinical murine model exposed to cigarette smoke, lower airway dysbiosis with common oral commensals augments the inflammatory injury, revealing transcriptomic signatures similar to those observed in human subjects with COPD. Conclusions: Lower airway dysbiosis in the setting of smoke exposure contributes to inflammatory injury early in COPD. Targeting the lower airway microbiome in combination with smoking cessation may be of potential therapeutic relevance.

Tyrosine Kinase Inhibitors Diminish Renal Neoplasms in a Tuberous Sclerosis Model Via Induction of Apoptosis.

Tuberous sclerosis complex (TSC) tumors are presently incurable despite a cytostatic response to mTOR pathway inhibition because recurrence of disease occurs after treatment is discontinued. Here, we explored the hypothesis that inhibiting tyrosine kinase activity in mesenchymal lineage-specific platelet-derived growth factor receptor ß (PDGFRß) signaling in TSC tumors is cytocidal and attenuates tumorigenesis at significantly higher levels than treatment with an mTOR inhibitor. Rapamycin-induced versus tyrosine kinase inhibitor (TKI)-induced renal angiomyolipoma (AML) and pulmonary lymphangioleiomyomatosis (LAM) tumor cells were comparatively analyzed using cell survival assays, RNA sequencing, and bioinformatics to distinguish tumoricidal mechanisms adopted by each drug type. The efficacy of imatinib therapy was validated against spontaneously developing renal cystadenomas in tuberous sclerosis Tsc2+/- mouse models (C57BL/6J mice; N = 6; 400 mg/kg/d; oral gavage) compared with Tsc2+/- mice treated with PBS (C57BL/6J mice; N = 6). Our study revealed that TKIs imatinib and nilotinib were cytocidal to both pulmonary LAM and renal AML cell cultures through the downregulation of the glycoprotein GPVI pathway and resultant disruption in mitochondrial permeability, increased cytosolic cytochrome C, and caspase 3 activation. Importantly, renal tumor growth was significantly attenuated in imatinib-treated Tsc2+/- mice compared with PBS treatment. The preclinical studies reported here provide evidence documenting the effectiveness of TKIs in limiting LAM and AML cell growth and viability with important clinical potential. Furthermore, these drugs elicit their effects by targeting a PDGF pathway-dependent apoptotic mechanism supporting the investigation of these drugs as a novel class of TSC therapeutics.

Blocking RAGE expression after injury reduces inflammation in mouse model of acute lung injury.

BACKGROUND: Receptor for Advanced Glycated Endproducts (RAGE) plays a major role in the inflammatory response to infectious and toxin induced acute lung injury. We tested the hypothesis that a RAGE blocking antibody when administered after the onset of injury can reduce lung inflammation compared to control antibody. METHODS: Male and female C57BL/6 (WT) mice were used. Forty-six received lipopolysaccharide (LPS) and 26 PBS by nasal instillation on day one, repeated on day three. On day 2, 36 mice receiving LPS were divided into two groups of 18, one treated with 200 µg of non-immune isotype control IgG and the second group treated with 200 µg of anti-RAGE Ab, each dose divided between IV and IP. Ten of the 46 were not treated. On day 4, before euthanasia, mice were injected with fluorescein isothiocyanate (FITC) labelled albumen. BALF and serum samples were collected as well as lung tissue for immunohistochemistry (IHC). BALF was analyzed for cell (leukocyte) counts, for FITC BALF/serum ratios indicating pulmonary vascular leak, and for cytokines/chemokines using bead based multiplex assays. Quantitative IHC was performed for MPO and RAGE. RESULTS: Ten LPS mice showed minimal inflammation by all measures indicating poor delivery of LPS and were excluded from analysis leaving n = 11 in the LPS + IgG group and n = 12 in the LPS + anti-RAGE group. BALF cell counts were low in the PBS administered mice (4.9 ± 2.1 × 105/ml) and high in the LPS injured untreated mice (109 ± 34) and in the LPS + IgG mice (91 ± 54) while in comparison, LPS + anti-RAGE ab mice counts were significantly lower (51.3 ± 18 vs. LPS + IgG, P = 0.03). The BALF/serum FITC ratios were lower for the LPS + anti-RAGE mice than for the LPS + IgG mice indicating less capillary leakiness. Quantitative IHC RAGE staining was lower in the LPS + anti-RAGE ab mice than in the LPS + IgG treated mice (P = 0.02). CONCLUSIONS: These results describe a four-day LPS protocol to sustain lung injury and allow for treatment and suggests that treatment aimed at blocking RAGE when given after onset of injury can reduce lung inflammation.

Consensus Statements on Deployment-Related Respiratory Disease, Inclusive of Constrictive Bronchiolitis: A Modified Delphi Study.

BACKGROUND: The diagnosis of constrictive bronchiolitis (CB) in previously deployed individuals, and evaluation of respiratory symptoms more broadly, presents considerable challenges, including using consistent histopathologic criteria and clinical assessments. RESEARCH QUESTION: What are the recommended diagnostic workup and associated terminology of respiratory symptoms in previously deployed individuals? STUDY DESIGN AND METHODS: Nineteen experts participated in a three-round modified Delphi study, ranking their level of agreement for each statement with an a priori definition of consensus. Additionally, rank-order voting on the recommended diagnostic approach and terminology was performed. RESULTS: Twenty-five of 28 statements reached consensus, including the definition of CB as a histologic pattern of lung injury that occurs in some previously deployed individuals while recognizing the importance of considering alternative diagnoses. Consensus statements also identified a diagnostic approach for the previously deployed individual with respiratory symptoms, distinguishing assessments best performed at a local or specialty referral center. Also, deployment-related respiratory disease (DRRD) was proposed as a broad term to subsume a wide range of potential syndromes and conditions identified through noninvasive evaluation or when surgical lung biopsy reveals evidence of multicompartmental lung injury that may include CB. INTERPRETATION: Using a modified Delphi technique, consensus statements provide a clinical approach to possible CB in previously deployed individuals. Use of DRRD provides a broad descriptor encompassing a range of postdeployment respiratory findings. Additional follow-up of individuals with DRRD is needed to assess disease progression and to define other features of its natural history, which could inform physicians better and lead to evolution in this nosology.

Wingless/integrase-1 signaling in allergic asthma and pediatric lung diseases.

PURPOSE OF REVIEW: To provide an update on the current understanding of the role of wingless/integrase-1 (Wnt) signaling in pediatric allergic asthma and other pediatric lung diseases. RECENT FINDINGS: The Wnt signaling pathway is critical for normal lung development. Genetic and epigenetic human studies indicate a link between Wnt signaling and the development and severity of asthma in children. Mechanistic studies using animal models of allergic asthma demonstrate a key role for Wnt signaling in allergic airway inflammation and remodeling. More recently, data on bronchopulmonary dysplasia (BPD) pathogenesis points to the Wnt signaling pathway as an important regulator. SUMMARY: Current data indicates that the Wnt signaling pathway is an important mediator in allergic asthma and BPD pathogenesis. Further studies are needed to characterize the roles of individual Wnt signals in childhood disease, and to identify potential novel therapeutic targets to slow or prevent disease processes.

The Critical Role of MMP13 in Regulating Tooth Development and Reactionary Dentinogenesis Repair Through the Wnt Signaling Pathway.

Matrix-metalloproteinase-13 (MMP13) is important for bone formation and remodeling; however, its role in tooth development remains unknown. To investigate this, MMP13-knockout (Mmp13 -/- ) mice were used to analyze phenotypic changes in the dentin-pulp complex, mineralization-associated marker-expression, and mechanistic interactions. Immunohistochemistry demonstrated high MMP13-expression in pulp-tissue, ameloblasts, odontoblasts, and dentin in developing WT-molars, which reduced in adults, with human-DPC cultures demonstrating a >2000-fold increase in Mmp13-expression during mineralization. Morphologically, Mmp13 -/- molars displayed critical alterations in the dentin-phenotype, affecting dentin-tubule regularity, the odontoblast-palisade and predentin-definition with significantly reduced dentin volume (∼30% incisor; 13% molar), and enamel and dentin mineral-density. Reactionary-tertiary-dentin in response to injury was reduced at Mmp13 -/- molar cusp-tips but with significantly more dystrophic pulpal mineralization in MMP13-null samples. Odontoblast differentiation-markers, nestin and DSP, reduced in expression after MMP13-loss in vivo, with reduced calcium deposition in MMP13-null DPC cultures. RNA-sequencing analysis of WT and Mmp13 -/- pulp highlighted 5,020 transcripts to have significantly >2.0-fold change, with pathway-analysis indicating downregulation of the Wnt-signaling pathway, supported by reduced in vivo expression of the Wnt-responsive gene Axin2. Mmp13 interaction with Axin2 could be partly responsible for the loss of odontoblastic activity and alteration to the tooth phenotype and volume which is evident in this study. Overall, our novel findings indicate MMP13 as critical for tooth development and mineralization processes, highlighting mechanistic interaction with the Wnt-signaling pathway.

Lung lymphatic thrombosis and dysfunction caused by cigarette smoke exposure precedes emphysema in mice.

The lymphatic vasculature is critical for lung function, but defects in lymphatic function in the pathogenesis of lung disease is understudied. In mice, lymphatic dysfunction alone is sufficient to cause lung injury that resembles human emphysema. Whether lymphatic function is disrupted in cigarette smoke (CS)-induced emphysema is unknown. In this study, we investigated the effect of CS on lung lymphatic function. Analysis of human lung tissue revealed significant lung lymphatic thrombosis in patients with emphysema compared to control smokers that increased with disease severity. In a mouse model, CS exposure led to lung lymphatic thrombosis, decreased lymphatic drainage, and impaired leukocyte trafficking that all preceded the development of emphysema. Proteomic analysis demonstrated an increased abundance of coagulation factors in the lymph draining from the lungs of CS-exposed mice compared to control mice. In addition, in vitro assays demonstrated a direct effect of CS on lymphatic endothelial cell integrity. These data show that CS exposure results in lung lymphatic dysfunction and a shift in thoracic lymph towards a prothrombic state. Furthermore, our data suggest that lymphatic dysfunction is due to effects of CS on the lymphatic vasculature that precede emphysema. These studies demonstrate a novel component of CS-induced lung injury that occurs early in the pathogenesis of emphysema.

Deaccelerated Myogenesis and Autophagy in Genetically Induced Pulmonary Emphysema.

Patients with chronic obstructive pulmonary disease (COPD)-pulmonary emphysema often develop locomotor muscle dysfunction, which entails reduced muscle mass and force-generation capacity and is associated with worse outcomes, including higher mortality. Myogenesis contributes to adult muscle integrity during injury-repair cycles. Injurious events crucially occur in the skeletal muscles of patients with COPD in the setting of exacerbations and infections, which lead to acute decompensations for limited periods of time, after which patients typically fail to recover the baseline status they had before the acute event. Autophagy, which is dysregulated in muscles from patients with COPD, is a key regulator of muscle stem-satellite- cells activation and myogenesis, yet very little research has so far mechanistically investigated the role of autophagy dysregulation in COPD muscles. Using a genetically inducible interleukin-13-driven pulmonary emphysema model leading to muscle dysfunction, and confirmed with a second genetic animal model, we found a significant myogenic dysfunction associated with the reduced proliferative capacity of satellite cells. Transplantation experiments followed by lineage tracing suggest that an intrinsic defect in satellite cells, and not in the COPD environment, plays a dominant role in the observed myogenic dysfunction. RNA sequencing analysis and direct observation of COPD mice satellite cells suggest dysregulated autophagy. Moreover, while autophagy flux experiments with bafilomycin demonstrated deacceleration of autophagosome turnover in COPD mice satellite cells, spermidine-induced autophagy stimulation leads to a higher replication rate and myogenesis in these animals. Our data suggest that pulmonary emphysema causes disrupted myogenesis, which could be improved with stimulation of autophagy and satellite cells activation, leading to an attenuated muscle dysfunction.

High mobility group AT-hook 2 regulates osteoblast differentiation and facial bone development.

The mutation and deletion of high mobility group AT-hook 2 (Hmga2) gene exhibit skeletal malformation, but almost nothing is known about the mechanism. This study examined morphological anomaly of facial bone in Hmga2-/- mice and osteoblast differentiation of pre-osteoblast MC3T3-E1 cells with Hmga2 gene knockout (A2KO). Hmga2-/- mice showed the size reduction of anterior frontal part of facial bones. Hmga2 protein and mRNA were expressed in mesenchymal cells at ossification area of nasal bone. A2KO cells differentiation into osteoblasts after reaching the proliferation plateau was strongly suppressed by alizarin red and alkaline phosphatase staining analyses. Expression of osteoblast-related genes, especially Osterix, was down-regulated in A2KO cells. These results demonstrate a close association of Hmga2 with osteoblast differentiation of mesenchymal cells and bone growth. Although future studies are needed, the present study suggests an involvement of Hmga2 in osteoblast-genesis and bone growth.

The Role of Secreted Frizzled-related Protein-1 in Allergic Asthma.

Although allergic asthma is a highly prevalent chronic inflammatory condition, the underlying pathogenesis driving T-helper cell type 2 inflammation is not well understood. Wnt/ß-catenin signaling has been implicated, but the influence of individual members of the pathway is not clear. We hypothesized that SFRP-1 (secreted frizzled-related protein-1), a Wnt signaling modulator, plays an important role in the development of allergic inflammation in asthma. Using an in vivo house dust mite asthma model, SFRP-1-/- mice were sensitized, and their BAL fluid was collected to evaluate airway inflammation. SFRP-1-/- mice exhibited less inflammation with reduced cellular infiltration and concentration of IL-5 in bronchoalveolar lavage fluid compared with wild-type (WT) mice. Similar findings were observed in WT mice treated with SFRP-1 inhibitor, WAY316606. Alveolar macrophages from sensitized SFRP-1-/- mice demonstrated reduced alternative polarization compared with WT, indicating that macrophages could mediate the alteration in inflammation seen in these mice. These findings suggest that SFRP-1 is an important potentiator of asthmatic airway inflammation.

Can lightning strike twice? Wild-type transthyretin cardiac amyloidosis associated with rare liver disease.

Wild-type ATTR cardiac amyloidosis (ATTRwt-CA) is not as rare as previously thought to be. Patients with infiltrative cardiac amyloidosis often present with right-sided heart failure (HF) symptomatology. Clinically significant liver disease and cirrhosis has not been reported in ATTRwt-CA. We present two cases of ATTRwt-CA with right-sided HF and abnormal liver function tests initially thought to be secondary to congestive hepatopathy but found to have rare and unrelated liver disease. These cases highlight the importance of developing a broad differential diagnosis and leveraging a multidisciplinary team approach in evaluating patients for unusual causes of cirrhosis/other chronic liver diseases when ATTR cardiac amyloidosis patients present with congestive hepatopathy.

WHotLAMP: A simple, inexpensive, and sensitive molecular test for the detection of SARS-CoV-2 in saliva.

Despite the development of effective vaccines against SARS-CoV-2, epidemiological control of the virus is still challenging due to slow vaccine rollouts, incomplete vaccine protection to current and emerging variants, and unwillingness to get vaccinated. Therefore, frequent testing of individuals to identify early SARS-CoV-2 infections, contact-tracing and isolation strategies remain crucial to mitigate viral spread. Here, we describe WHotLAMP, a rapid molecular test to detect SARS-CoV-2 in saliva. WHotLAMP is simple to use, highly sensitive (~4 viral particles per microliter of saliva) and specific, as well as inexpensive, making it ideal for frequent screening. Moreover, WHotLAMP does not require toxic chemicals or specialized equipment and thus can be performed in point-of-care settings, and may also be adapted for resource-limited environments or home use. While applied here to SARS-CoV-2, WHotLAMP can be modified to detect other pathogens, making it adaptable for other diagnostic assays, including for use in future outbreaks.

Renal neoplasms in tuberous sclerosis mice are neurocristopathies.

Tuberous sclerosis (TS) is a rare disorder exhibiting multi-systemic benign neoplasms. We hypothesized the origin of TS neoplastic cells derived from the neural crest given the heterogeneous ecto-mesenchymal phenotype of the most common TS neoplasms. To test this hypothesis, we employed Cre-loxP lineage tracing of myelin protein zero (Mpz)-expressing neural crest cells (NCCs) in spontaneously developing renal tumors of Tsc2 +/- /Mpz(Cre)/TdT fl/fl reporter mice. In these mice, ectopic renal tumor onset was detected at 4 months of age increasing in volume by 16 months of age with concomitant increase in the subpopulation of tdTomato+ NCCs from 0% to 6.45% of the total number of renal tumor cells. Our results suggest that Tsc2 +/- mouse renal tumors arise from domiciled proliferative progenitor cell populations of neural crest origin that co-opt tumorigenesis due to mutations in Tsc2 loci. Targeting neural crest antigenic determinants will provide a potential alternative therapeutic approach for TS pathogenesis.

Integrated transcriptomic analysis of human tuberculosis granulomas and a biomimetic model identifies therapeutic targets.

Tuberculosis (TB) is a persistent global pandemic, and standard treatment for it has not changed for 30 years. Mycobacterium tuberculosis (Mtb) has undergone prolonged coevolution with humans, and patients can control Mtb even after extensive infection, demonstrating the fine balance between protective and pathological host responses within infected granulomas. We hypothesized that whole transcriptome analysis of human TB granulomas isolated by laser capture microdissection could identify therapeutic targets, and that comparison with a noninfectious granulomatous disease, sarcoidosis, would identify disease-specific pathological mechanisms. Bioinformatic analysis of RNAseq data identified numerous shared pathways between TB and sarcoidosis lymph nodes, and also specific clusters demonstrating TB results from a dysregulated inflammatory immune response. To translate these insights, we compared 3 primary human cell culture models at the whole transcriptome level and demonstrated that the 3D collagen granuloma model most closely reflected human TB disease. We investigated shared signaling pathways with human disease and identified 12 intracellular enzymes as potential therapeutic targets. Sphingosine kinase 1 inhibition controlled Mtb growth, concurrently reducing intracellular pH in infected monocytes and suppressing inflammatory mediator secretion. Immunohistochemical staining confirmed that sphingosine kinase 1 is expressed in human lung TB granulomas, and therefore represents a host therapeutic target to improve TB outcomes.

Knockdown of Alpha-1 Antitrypsin with antisense oligonucleotide does not exacerbate smoke induced lung injury.

Alpha-1 Antitrypsin (AAT) is a serum protease inhibitor that regulates increased lung protease production induced by cigarette smoking. Mutations in the Serpina1 gene cause AAT to form hepatoxic polymers, which can lead to reduced availability for the protein's primary function and severe liver disease. An AAT antisense oligonucleotide (ASO) was previously identified to be beneficial for the AATD liver disease by blocking the mutated AAT transcripts. Here we hypothesized that knockdown of AAT aggravates murine lung injury during smoke exposure and acute exacerbations of chronic obstructive pulmonary disease (COPD). C57BL/6J mice were randomly divided into 4 groups each for the smoking and smoke-flu injury models. The ASO and control (No-ASO) were injected subcutaneously starting with smoking or four days prior to influenza infection and then injected weekly at 50 mg/kg body weight. ASO treatment during a 3-month smoke exposure significantly decreased the serum and lung AAT expression, resulting in increased Cela1 expression and elastase activity. However, despite the decrease in AAT, neither the inflammatory cell counts in the bronchoalveolar lavage fluid (BALF) nor the lung structural changes were significantly worsened by ASO treatment. We observed significant differences in inflammation and emphysema due to smoke exposure, but did not observe an ASO treatment effect. Similarly, with the smoke-flu model, differences were only observed between smoke-flu and room air controls, but not as a result of ASO treatment. Off-target effects or compensatory mechanisms may account for this finding. Alternatively, the reduction of AAT with ASO treatment, while sufficient to protect from liver injury, may not be robust enough to lead to lung injury. The results also suggest that previously described AAT ASO treatment for AAT mutation related liver disease may attenuate hepatic injury without being detrimental to the lungs. These potential mechanisms need to be further investigated in order to fully understand the impact of AAT inhibition on protease-antiprotease imbalance in the murine smoke exposure model.

Episodic Aspiration with Oral Commensals Induces a MyD88-dependent, Pulmonary T-Helper Cell Type 17 Response that Mitigates Susceptibility to Streptococcus pneumoniae.

Rationale: Cross-sectional human data suggest that enrichment of oral anaerobic bacteria in the lung is associated with an increased T-helper cell type 17 (Th17) inflammatory phenotype.Objectives: In this study, we evaluated the microbial and host immune-response dynamics after aspiration with oral commensals using a preclinical mouse model.Methods: Aspiration with a mixture of human oral commensals (MOC; Prevotella melaninogenica, Veillonella parvula, and Streptococcus mitis) was modeled in mice followed by variable time of killing. The genetic backgrounds of mice included wild-type, MyD88-knockout, and STAT3C backgrounds.Measurements and Main Results: 16S-rRNA gene sequencing characterized changes in microbiota. Flow cytometry, cytokine measurement via Luminex and RNA host-transcriptome sequencing was used to characterize the host immune phenotype. Although MOC aspiration correlated with lower-airway dysbiosis that resolved within 5 days, it induced an extended inflammatory response associated with IL-17-producing T cells lasting at least 14 days. MyD88 expression was required for the IL-17 response to MOC aspiration, but not for T-cell activation or IFN-γ expression. MOC aspiration before a respiratory challenge with S. pneumoniae led to a decrease in hosts' susceptibility to this pathogen.Conclusions: Thus, in otherwise healthy mice, a single aspiration event with oral commensals is rapidly cleared from the lower airways but induces a prolonged Th17 response that secondarily decreases susceptibility to S. pneumoniae. Translationally, these data implicate an immunoprotective role of episodic microaspiration of oral microbes in the regulation of the lung immune phenotype and mitigation of host susceptibility to infection with lower-airway pathogens.

Attenuation of pulmonary injury by an inhaled MMP inhibitor in the endotoxin lung injury model.

Acute respiratory distress syndrome (ARDS) is characterized by pulmonary edema and poor gas exchange resulting from severe inflammatory lung injury. Neutrophilic infiltration and increased pulmonary vascular permeability are hallmarks of early ARDS and precipitate a self-perpetuating cascade of inflammatory signaling. The biochemical processes initiating these events remain unclear. Typically associated with extracellular matrix degradation, recent data suggest matrix metalloproteinases (MMPs) are regulators of pulmonary inflammation. To demonstrate that inhalation of a broad MMP inhibitor attenuates LPS induced pulmonary inflammation. Nebulized CGS27023AM (CGS) was administered to LPS-injured mice. Pulmonary CGS levels were examined by mass spectroscopy. Inflammatory scoring of hematoxylin-eosin sections, examination of vascular integrity via lung wet/dry and bronchoalveolar lvage/serum FITC-albumin ratios were performed. Cleaved caspase-3 levels were also assessed. Differential cell counts and pulse-chase labeling were utilized to determine the effects of CGS on neutrophil migration. The effects of CGS on human neutrophil migration and viability were examined using Boyden chambers and MTT assays. Nebulization successfully delivered CGS to the lungs. Treatment decreased pulmonary inflammatory scores, edema, and apoptosis in LPS treated animals. Neutrophil chemotaxis was reduced by CGS treatment, with inhalation causing significant reductions in both the total number and newly produced bromodeoxyuridine-positive cells infiltrating the lung. Mechanistic studies on cells isolated from humans demonstrate that CGS-treated neutrophils exhibit decreased chemotaxis. The protective effect observed following treatment with a nonspecific MMP inhibitor indicates that one or more MMPs mediate the development of pulmonary edema and neutrophil infiltration in response to LPS injury. In accordance with this, inhaled MMP inhibitors warrant further study as a potential new therapeutic avenue for treatment of acute lung injury.