Human Pluripotency Is Initiated and Preserved by a Unique Subset of Founder Cells.

The assembly of organized colonies is the earliest manifestation in the derivation or induction of pluripotency in vitro. However, the necessity and origin of this assemblance is unknown. Here, we identify human pluripotent founder cells (hPFCs) that initiate, as well as preserve and establish, pluripotent stem cell (PSC) cultures. PFCs are marked by N-cadherin expression (NCAD+) and reside exclusively at the colony boundary of primate PSCs. As demonstrated by functional analysis, hPFCs harbor the clonogenic capacity of PSC cultures and emerge prior to commitment events or phenotypes associated with pluripotent reprogramming. Comparative single-cell analysis with pre- and post-implantation primate embryos revealed hPFCs share hallmark properties with primitive endoderm (PrE) and can be regulated by non-canonical Wnt signaling. Uniquely informed by primate embryo organization in vivo, our study defines a subset of founder cells critical to the establishment pluripotent state.

Hexokinase Is an Innate Immune Receptor for the Detection of Bacterial Peptidoglycan.

Degradation of Gram-positive bacterial cell wall peptidoglycan in macrophage and dendritic cell phagosomes leads to activation of the NLRP3 inflammasome, a cytosolic complex that regulates processing and secretion of interleukin (IL)-1ß and IL-18. While many inflammatory responses to peptidoglycan are mediated by detection of its muramyl dipeptide component in the cytosol by NOD2, we report here that NLRP3 inflammasome activation is caused by release of N-acetylglucosamine that is detected in the cytosol by the glycolytic enzyme hexokinase. Inhibition of hexokinase by N-acetylglucosamine causes its dissociation from mitochondria outer membranes, and we found that this is sufficient to activate the NLRP3 inflammasome. In addition, we observed that glycolytic inhibitors and metabolic conditions affecting hexokinase function and localization induce inflammasome activation. While previous studies have demonstrated that signaling by pattern recognition receptors can regulate metabolic processes, this study shows that a metabolic enzyme can act as a pattern recognition receptor. PAPERCLIP.

Regulation of ferroptotic cancer cell death by GPX4.

Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosis-inducing small molecules. We used targeted metabolomic profiling to discover that depletion of glutathione causes inactivation of glutathione peroxidases (GPXs) in response to one class of compounds and a chemoproteomics strategy to discover that GPX4 is directly inhibited by a second class of compounds. GPX4 overexpression and knockdown modulated the lethality of 12 ferroptosis inducers, but not of 11 compounds with other lethal mechanisms. In addition, two representative ferroptosis inducers prevented tumor growth in xenograft mouse tumor models. Sensitivity profiling in 177 cancer cell lines revealed that diffuse large B cell lymphomas and renal cell carcinomas are particularly susceptible to GPX4-regulated ferroptosis. Thus, GPX4 is an essential regulator of ferroptotic cancer cell death.

Intestinal Permeability and IgA Provoke Immune Vasculitis Linked to Cardiovascular Inflammation.

Recent experimental data and clinical, genetic, and transcriptome evidence from patients converge to suggest a key role of interleukin-1ß (IL-1ß) in the pathogenesis of Kawasaki disease (KD). However, the molecular mechanisms involved in the development of cardiovascular lesions during KD vasculitis are still unknown. Here, we investigated intestinal barrier function in KD vasculitis and observed evidence of intestinal permeability and elevated circulating secretory immunoglobulin A (sIgA) in KD patients, as well as elevated sIgA and IgA deposition in vascular tissues in a mouse model of KD vasculitis. Targeting intestinal permeability corrected gut permeability, prevented IgA deposition and ameliorated cardiovascular pathology in the mouse model. Using genetic and pharmacologic inhibition of IL-1ß signaling, we demonstrate that IL-1ß lies upstream of disrupted intestinal barrier function, subsequent IgA vasculitis development, and cardiac inflammation. Targeting mucosal barrier dysfunction and the IL-1ß pathway may also be applicable to other IgA-related diseases, including IgA vasculitis and IgA nephropathy.

T-Cell-Intrinsic Receptor Interacting Protein 2 Regulates Pathogenic T Helper 17 Cell Differentiation.

Receptor interacting protein 2 (RIP2) plays a role in sensing intracellular pathogens, but its function in T cells is unclear. We show that RIP2 deficiency in CD4+ T cells resulted in chronic and severe interleukin-17A-mediated inflammation during Chlamydia pneumoniae lung infection, increased T helper 17 (Th17) cell formation in lungs of infected mice, accelerated atherosclerosis, and more severe experimental autoimmune encephalomyelitis. While RIP2 deficiency resulted in reduced conventional Th17 cell differentiation, it led to significantly enhanced differentiation of pathogenic (p)Th17 cells, which was dependent on RORα transcription factor and interleukin-1 but independent of nucleotide oligomerization domain (NOD) 1 and 2. Overexpression of RIP2 resulted in suppression of pTh17 cell differentiation, an effect mediated by its CARD domain, and phenocopied by a cell-permeable RIP2 CARD peptide. Our data suggest that RIP2 has a T cell-intrinsic role in determining the balance between homeostatic and pathogenic Th17 cell responses.

POPsicle for Fever! Cooling Down the Inflammasome.

Inhibition of the inflammasome might be beneficial for numerous inflammatory pathologies. In this issue of Immunity, de Almeida et al. (2015) report that the PYRIN domain-only protein (POP1) efficiently inhibits inflammasome activation, identifying it as a pan-inflammasome inhibitor.

Lipopolysaccharide Induces Alveolar Macrophage Necrosis via CD14 and the P2X7 Receptor Leading to Interleukin-1α Release.

Acute lung injury (ALI) remains a serious health issue with little improvement in our understanding of the pathophysiology and therapeutic approaches. We investigated the mechanism that lipopolysaccharide (LPS) induces early neutrophil recruitment to lungs and increases pulmonary vascular permeability during ALI. Intratracheal LPS induced release of pro-interleukin-1α (IL-1α) from necrotic alveolar macrophages (AM), which activated endothelial cells (EC) to induce vascular leakage via loss of vascular endothelial (VE)-cadherin. LPS triggered the AM purinergic receptor P2X7(R) to induce Ca(2+) influx and ATP depletion, which led to necrosis. P2X7R deficiency significantly reduced necrotic death of AM and release of pro-IL-1α into the lung. CD14 was required for LPS binding to P2X7R, as CD14 neutralization significantly diminished LPS induced necrotic death of AM and pro-IL-1α release. These results demonstrate a key role for pro-IL-1α from necrotic alveolar macrophages in LPS-mediated ALI, as a critical initiator of increased vascular permeability and early neutrophil infiltration.

NLRP3 Inflammasome Mediates Immune-Stromal Interactions in Vasculitis.

[Figure: see text].

Dependency of a therapy-resistant state of cancer cells on a lipid peroxidase pathway.

Plasticity of the cell state has been proposed to drive resistance to multiple classes of cancer therapies, thereby limiting their effectiveness. A high-mesenchymal cell state observed in human tumours and cancer cell lines has been associated with resistance to multiple treatment modalities across diverse cancer lineages, but the mechanistic underpinning for this state has remained incompletely understood. Here we molecularly characterize this therapy-resistant high-mesenchymal cell state in human cancer cell lines and organoids and show that it depends on a druggable lipid-peroxidase pathway that protects against ferroptosis, a non-apoptotic form of cell death induced by the build-up of toxic lipid peroxides. We show that this cell state is characterized by activity of enzymes that promote the synthesis of polyunsaturated lipids. These lipids are the substrates for lipid peroxidation by lipoxygenase enzymes. This lipid metabolism creates a dependency on pathways converging on the phospholipid glutathione peroxidase (GPX4), a selenocysteine-containing enzyme that dissipates lipid peroxides and thereby prevents the iron-mediated reactions of peroxides that induce ferroptotic cell death. Dependency on GPX4 was found to exist across diverse therapy-resistant states characterized by high expression of ZEB1, including epithelial-mesenchymal transition in epithelial-derived carcinomas, TGFß-mediated therapy-resistance in melanoma, treatment-induced neuroendocrine transdifferentiation in prostate cancer, and sarcomas, which are fixed in a mesenchymal state owing to their cells of origin. We identify vulnerability to ferroptic cell death induced by inhibition of a lipid peroxidase pathway as a feature of therapy-resistant cancer cells across diverse mesenchymal cell-state contexts.

Overexpressed angiotensin-converting enzyme in neutrophils suppresses glomerular damage in crescentic glomerulonephritis.

While angiotensin-converting enzyme (ACE) regulates blood pressure by producing angiotensin II as part of the renin-angiotensin system, we recently reported that elevated ACE in neutrophils promotes an effective immune response and increases resistance to infection. Here, we investigate if such neutrophils protect against renal injury in immune complex (IC)-mediated crescentic glomerulonephritis (GN) through complement. Nephrotoxic serum nephritis (NTN) was induced in wild-type and NeuACE mice that overexpress ACE in neutrophils. Glomerular injury of NTN in NeuACE mice was attenuated with much less proteinuria, milder histological injury, and reduced IC deposits, but presented with more glomerular neutrophils in the early stage of the disease. There were no significant defects in T and B cell functions in NeuACE mice. NeuACE neutrophils exhibited enhanced IC uptake with elevated surface expression of FcγRII/III and complement receptor CR1/2. IC uptake in neutrophils was enhanced by NeuACE serum containing elevated complement C3b. Given no significant complement activation by ACE, this suggests that neutrophil ACE indirectly preactivates C3 and that the C3b-CR1/2 axis and elevated FcγRII/III play a central role in IC elimination by neutrophils, resulting in reduced glomerular injury. The present study identified a novel renoprotective role of ACE in glomerulonephritis; elevated neutrophilic ACE promotes elimination of locally formed ICs in glomeruli via C3b-CR1/2 and FcγRII/III, ameliorating glomerular injury.NEW & NOTEWORTHY We studied immune complex (IC)-mediated crescentic glomerulonephritis in NeuACE mice that overexpress ACE only in neutrophils. Such mice show no significant defects in humoral immunity but strongly resist nephrotoxic serum nephritis (less proteinuria, milder histological damage, reduced IC deposits, and more glomerular neutrophils). NeuACE neutrophils enhanced IC uptake via increased surface expression of CR1/2 and FcgRII/III, as well as elevated serum complement C3b. These results suggest neutrophil ACE as a novel approach to reducing glomerulonephritis.

Nationwide time-series surveys of pressure ulcer prevalence in Japan.

OBJECTIVE: The Japanese Society of Pressure Ulcers (JSPU) has two purposes: first, to improve knowledge and skills among health professionals related to preventing and managing pressure ulcers (PUs); and second, to represent those in the field managing PUs, including with government and health authorities. Since 2006, JSPU has conducted fact-finding surveys about every four years to identify PU prevalence in Japan (2006, 2010, 2013 and 2016). Based on the prevalence identified by these surveys, an attempt was made to validate the achievements of JSPU's activities. METHOD: Information from one-day surveys of hospitals, long-term care health facilities, long-term care welfare facilities, and home visit nursing care stations was analysed. We used generalised estimating equations to estimate the proportions of PUs and their 95% confidence intervals (CIs) for each survey. RESULTS: A total of 662,419 patients in 2631 facilities participated in the surveys. The estimated proportions for all facilities (95% CI) in chronological order, from the first to the fourth survey, were: 2.67% (2.52-2.83); 2.61% (2.43-2.80); 1.99% (1.83-2.17); and 1.79% (1.65-1.94), respectively. In all facility types, the proportion of PUs was lower in the fourth survey than the first survey. CONCLUSION: The proportion of PUs showed a decreasing trend and was low according to global standards, demonstrating the efficacy of JSPU's activities.

Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis.

We report that in the presence of signal 1 (NF-κB), the NLRP3 inflammasome was activated by mitochondrial apoptotic signaling that licensed production of interleukin-1ß (IL-1ß). NLRP3 secondary signal activators such as ATP induced mitochondrial dysfunction and apoptosis, resulting in release of oxidized mitochondrial DNA (mtDNA) into the cytosol, where it bound to and activated the NLRP3 inflammasome. The antiapoptotic protein Bcl-2 inversely regulated mitochondrial dysfunction and NLRP3 inflammasome activation. Mitochondrial DNA directly induced NLRP3 inflammasome activation, because macrophages lacking mtDNA had severely attenuated IL-1ß production, yet still underwent apoptosis. Both binding of oxidized mtDNA to the NLRP3 inflammasome and IL-1ß secretion could be competitively inhibited by the oxidized nucleoside 8-OH-dG. Thus, our data reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome. These results provide a missing link between apoptosis and inflammasome activation, via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome.

Unsupervised identification of disease states from high-dimensional physiological and histopathological profiles.

The liver and kidney in mammals play central roles in protecting the organism from xenobiotics and are at high risk of xenobiotic-induced injury. Xenobiotic-induced tissue injury has been extensively studied from both classical histopathological and biochemical perspectives. Here, we introduce a machine-learning approach to analyze toxicological response. Unsupervised characterization of physiological and histological changes in a large toxicogenomic dataset revealed nine discrete toxin-induced disease states, some of which correspond to known pathology, but others were novel. Analysis of dynamics revealed transitions between disease states at constant toxin exposure, mostly toward decreased pathology, implying induction of tolerance. Tolerance correlated with induction of known xenobiotic defense genes and decrease of novel ferroptosis sensitivity biomarkers, suggesting ferroptosis as a druggable driver of tissue pathophysiology. Lastly, mechanism of body weight decrease, a known primary marker for xenobiotic toxicity, was investigated. Combined analysis of food consumption, body weight, and molecular biomarkers indicated that organ injury promotes cachexia by whole-body signaling through Gdf15 and Igf1, suggesting strategies for therapeutic intervention that may be broadly relevant to human disease.

Global survey of cell death mechanisms reveals metabolic regulation of ferroptosis.

Apoptosis is one type of programmed cell death. Increasingly, non-apoptotic cell death is recognized as being genetically controlled, or 'regulated'. However, the full extent and diversity of alternative cell death mechanisms remain uncharted. Here we surveyed the landscape of pharmacologically accessible cell death mechanisms. In an examination of 56 caspase-independent lethal compounds, modulatory profiling showed that 10 compounds induced three different types of regulated non-apoptotic cell death. Optimization of one of those ten resulted in the discovery of FIN56, a specific inducer of ferroptosis. Ferroptosis has been found to occur when the lipid-repair enzyme GPX4 is inhibited. FIN56 promoted degradation of GPX4. FIN56 also bound to and activated squalene synthase, an enzyme involved in isoprenoid biosynthesis, independent of GPX4 degradation. These discoveries show that dysregulation of lipid metabolism is associated with ferroptosis. This systematic approach is a means to discover and characterize novel cell death phenotypes.

Ogg1-Dependent DNA Repair Regulates NLRP3 Inflammasome and Prevents Atherosclerosis.

RATIONALE: Activation of NLRP3 (nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3) inflammasome-mediating interleukin (IL)-1ß secretion has emerged as an important component of inflammatory processes in atherosclerosis. Mitochondrial DNA (mtDNA) damage is detrimental in atherosclerosis, and mitochondria are central regulators of the nucleotide-binding domain and leucine-rich repeat pyrin domain containing 3 inflammasome. Human atherosclerotic plaques express increased mtDNA damage. The major DNA glycosylase, 8-oxoguanine glycosylase (OGG1), is responsible for removing the most abundant form of oxidative DNA damage. OBJECTIVE: To test the role of OGG1 in the development of atherosclerosis in mouse. METHODS AND RESULTS: We observed that Ogg1 expression decreases over time in atherosclerotic lesion macrophages of low-density lipoprotein receptor (Ldlr) knockout mice fed a Western diet. Ogg1(-/-)Ldlr(-/-) mice fed a Western diet resulted in an increase in plaque size and lipid content. We found increased oxidized mtDNA, inflammasome activation, and apoptosis in atherosclerotic lesions and also higher serum IL-1ß and IL-18 in Ogg1(-/-)Ldlr(-/-) mice than in Ldlr(-/-). Transplantation with Ogg1(-/-) bone marrow into Ldlr(-/-) mice led to larger atherosclerotic lesions and increased IL-1ß production. However, transplantation of Ogg1(-/-)Nlrp3(-/-) bone marrow reversed the Ogg1(-/-) phenotype of increased plaque size. Ogg1(-/-) macrophages showed increased oxidized mtDNA and had greater amounts of cytosolic mtDNA and cytochrome c, increased apoptosis, and more IL-1ß secretion. Finally, we found that proatherogenic miR-33 can directly inhibit human OGG1 expression and indirectly suppress both mouse and human OGG1 via AMP-activated protein kinase. CONCLUSIONS: OGG1 plays a protective role in atherogenesis by preventing excessive inflammasome activation. Our study provides insight into a new target for therapeutic intervention based on a link between oxidative mtDNA damage, OGG1, and atherosclerosis via NLRP3 inflammasome.

The Japanese registry for surgery of ischial pressure ulcers: STANDARDS-I.

OBJECTIVE: To clarify the surgical indications and the appropriate perioperative management of ischial pressure ulcers (PUs). METHOD: A two-year prospective, nationwide registry study was carried out across 26 medical institutions in Japan. All participating institutions managed ischial PUs according to the standardisation of total management and surgical application for the refractory decubitus (STANDARDS-I) perioperative protocol. Analysis was conducted on a range of clinically or statistically important variables for the achievement of primary or secondary endpoints: complete wound healing and hospital discharge at three months, and complete wound healing at one month after surgery, respectively. RESULTS: A total of 59 patients took part in the study. All patients underwent surgery for ischial PUs during the study period. Patients who had achieved the primary endpoint had a higer preoperative functional independence measurement (FIM score), a higher 'G' score in the DESIGN-R scale and were more likely to have healed by primary intention. Patients who had achieved the secondary endpoint were more likely to have spastic paralysis, preoperative physiotherapy and localised infection of the wound, among other variables. CONCLUSION: This survey suggests that preoperative physiotherapy increases the speed of wound healing, and good granulation of the wound bed preoperatively increases the likelihood of woundless discharge from hospital, whereas the existence of comorbidities negatively influences the likelihood of woundless discharge from hospital. The study also suggests that the existence of spastic paralysis, preoperative infection of the wound, or surgical reduction of the ischial tubercle speeds up the healing of the wound. However, the wound failed to heal significantly more often in patients with increasing white blood cell count after surgery.

The clinical impact of edoxaban for the patients with postoperative anemia after total hip arthroplasty.

BACKGROUND: Postoperative anemia is a common complication after total hip arthroplasty (THA). However, the effect of edoxaban on postoperative anemia after THA remains unclear. Here, we retrospectively evaluated the clinical assessment of postoperative anemia and the associated changes of coagulation parameters in patients undergoing thromboprophylaxis with edoxaban compared with fondaparinux as a conventional anticoagulant thromboprophylactic agent after THA. METHODS: One hundred and forty-nine patients who underwent THA from July 2010 to June 2012 were divided into two groups, according to whether they were operated on before or after the approval of edoxaban: the fondaparinux group (Group F: 86 patients) and the edoxaban group (Group E: 63 patients). The frequency of postoperative anemia and blood coagulation values were investigated. RESULTS: Postoperative anemia developed more frequently in Group E than in Group F after surgery. However, the degree of postoperative anemia showed no significant difference between the groups. Meanwhile, prothrombin time (PT), prothrombin time-international normalized ratio (PT-INR), and activated partial thromboplastin time were markedly higher in patients with edoxaban-associated postoperative anemia, which showed an increased potential to predict the occurrence of postoperative anemia. Additionally, both PT and PT-INR in Group E were also correlated with the volume of estimated blood loss. CONCLUSION: The frequency of postoperative anemia was increased in patients treated with edoxaban, compared to fondaparinux, after THA. Edoxaban thromboprophylaxis might, therefore, require more careful monitoring to prevent postoperative anemia. Additionally, particular prolongation of PT and PT-INR induced by edoxaban treatment might predict postoperative anemia.

Role of Interleukin-1 Signaling in a Mouse Model of Kawasaki Disease-Associated Abdominal Aortic Aneurysm.

OBJECTIVE: Kawasaki disease (KD) is the most common cause of acquired cardiac disease in US children. In addition to coronary artery abnormalities and aneurysms, it can be associated with systemic arterial aneurysms. We evaluated the development of systemic arterial dilatation and aneurysms, including abdominal aortic aneurysm (AAA) in the Lactobacillus casei cell-wall extract (LCWE)-induced KD vasculitis mouse model. METHODS AND RESULTS: We discovered that in addition to aortitis, coronary arteritis and myocarditis, the LCWE-induced KD mouse model is also associated with abdominal aorta dilatation and AAA, as well as renal and iliac artery aneurysms. AAA induced in KD mice was exclusively infrarenal, both fusiform and saccular, with intimal proliferation, myofibroblastic proliferation, break in the elastin layer, vascular smooth muscle cell loss, and inflammatory cell accumulation in the media and adventitia. Il1r(-/-), Il1a(-/-), and Il1b(-/-) mice were protected from KD associated AAA. Infiltrating CD11c(+) macrophages produced active caspase-1, and caspase-1 or NLRP3 deficiency inhibited AAA formation. Treatment with interleukin (IL)-1R antagonist (Anakinra), anti-IL-1α, or anti-IL-1ß mAb blocked LCWE-induced AAA formation. CONCLUSIONS: Similar to clinical KD, the LCWE-induced KD vasculitis mouse model can also be accompanied by AAA formation. Both IL-1α and IL-1ß play a key role, and use of an IL-1R blocking agent that inhibits both pathways may be a promising therapeutic target not only for KD coronary arteritis, but also for the other systemic arterial aneurysms including AAA that maybe seen in severe cases of KD. The LCWE-induced vasculitis model may also represent an alternative model for AAA disease.

Alternatively spliced myeloid differentiation protein-2 inhibits TLR4-mediated lung inflammation.

We previously identified a novel alternatively spliced isoform of human myeloid differentiation protein-2 (MD-2s) that competitively inhibits binding of MD-2 to TLR4 in vitro. In this study, we investigated the protective role of MD-2s in LPS-induced acute lung injury by delivering intratracheally an adenovirus construct that expressed MD-2s (Ad-MD-2s). After adenovirus-mediated gene transfer, MD-2s was strongly expressed in lung epithelial cells and readily detected in bronchoalveolar lavage fluid. Compared to adenovirus serotype 5 containing an empty vector lacking a transgene control mice, Ad-MD-2s delivery resulted in significantly less LPS-induced inflammation in the lungs, including less protein leakage, cell recruitment, and expression of proinflammatory cytokines and chemokines, such as IL-6, keratinocyte chemoattractant, and MIP-2. Bronchoalveolar lavage fluid from Ad-MD-2s mice transferred into lungs of naive mice before intratracheal LPS challenge diminished proinflammatory cytokine levels. As house dust mite (HDM) sensitization is dependent on TLR4 and HDM Der p 2, a structural homolog of MD-2, we also investigated the effect of MD-2s on HDM-induced allergic airway inflammation. Ad-MD-2s given before HDM sensitization significantly inhibited subsequent allergic airway inflammation after HDM challenge, including reductions in eosinophils, goblet cell hyperplasia, and IL-5 levels. Our study indicates that the alternatively spliced short isoform of human MD-2 could be a potential therapeutic candidate to treat human diseases induced or exacerbated by TLR4 signaling, such as Gram-negative bacterial endotoxin-induced lung injury and HDM-triggered allergic lung inflammation.

Mast cells play an important role in chlamydia pneumoniae lung infection by facilitating immune cell recruitment into the airway.

Mast cells are known as central players in allergy and anaphylaxis, and they play a pivotal role in host defense against certain pathogens. Chlamydia pneumoniae is an important human pathogen, but it is unclear what role mast cells play during C. pneumoniae infection. We infected C57BL/6 (wild-type [WT]) and mast cell-deficient mice (Kit(W-sh/W-sh) [Wsh]) with C. pneumoniae. Wsh mice showed improved survival compared with WT mice, with fewer cells in Wsh bronchoalveolar lavage fluid (BALF), despite similar levels of cytokines and chemokines. We also found a more rapid clearance of bacteria from the lungs of Wsh mice compared with WT mice. Cromolyn, a mast cell stabilizer, reduced BALF cells and bacterial burden similar to the levels seen in Wsh mice; conversely, Compound 48/80, a mast cell degranulator, increased the number of BALF cells and bacterial burden. Histology showed that WT lungs had diffuse inflammation, whereas Wsh mice had patchy accumulations of neutrophils and perivascular accumulations of lymphocytes. Infected Wsh mice had reduced amounts of matrix metalloprotease-9 in BALF and were resistant to epithelial integral membrane protein degradation, suggesting that barrier integrity remains intact in Wsh mice. Mast cell reconstitution in Wsh mice led to enhanced bacterial growth and normal epithelial integral membrane protein degradation, highlighting the specific role of mast cells in this model. These data suggest that mast cells play a detrimental role during C. pneumoniae infection by facilitating immune cell infiltration into the airspace and providing a more favorable replicative environment for C. pneumoniae.