Biofilm exopolysaccharides alter sensory-neuron-mediated sickness during lung infection.

Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1+ sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness.

ARDS metabolic fingerprints: characterization, benchmarking, and potential mechanistic interpretation.

In this study, we aimed to identify acute respiratory distress syndrome (ARDS) metabolic fingerprints in selected patient cohorts and compare the metabolic profiles of direct versus indirect ARDS and hypoinflammatory versus hyperinflammatory ARDS. We hypothesized that the biological and inflammatory processes in ARDS would manifest as unique metabolomic fingerprints that set ARDS apart from other intensive care unit (ICU) conditions and could help examine ARDS subphenotypes and clinical subgroups. Patients with ARDS (n = 108) and ICU ventilated controls (n = 27) were included. Samples were randomly divided into 2/3 training and 1/3 test sets. Samples were analyzed using 1H nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. Twelve proteins/cytokines were also measured. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to select the most differentiating ARDS metabolites and protein/cytokines. Predictive performance of OPLS-DA models was measured in the test set. Temporal changes of metabolites were examined as patients progressed through ARDS until clinical recovery. Metabolic profiles of direct versus indirect ARDS subgroups and hypoinflammatory versus hyperinflammatory ARDS subgroups were compared. Serum metabolomics and proteins/cytokines had similar area under receiver operator curves when distinguishing ARDS from ICU controls. Pathway analysis of ARDS differentiating metabolites identified a dominant involvement of serine-glycine metabolism. In longitudinal tracking, the identified pathway metabolites generally exhibited correction by 7-14 days, coinciding with clinical improvement. ARDS subphenotypes and clinical subgroups were metabolically distinct. However, our identified metabolic fingerprints are not ARDS diagnostic biomarkers, and further research is required to ascertain generalizability. In conclusion, patients with ARDS are metabolically different from ICU controls. ARDS subphenotypes and clinical subgroups are metabolically distinct.

Fer kinase limits neutrophil chemotaxis toward end target chemoattractants.

Neutrophil recruitment and directional movement toward chemotactic stimuli are important processes in innate immune responses. This study examines the role of Fer kinase in neutrophil recruitment and chemotaxis to various chemoattractants in vitro and in vivo. Mice targeted with a kinase-inactivating mutation (Fer(DR/DR)) or wild type (WT) were studied using time-lapse intravital microscopy to examine leukocyte recruitment and chemotaxis in vivo. In response to keratinocyte-derived cytokine, no difference in leukocyte chemotaxis was observed between WT and Fer(DR/DR) mice. However, in response to the chemotactic peptide WKYMVm, a selective agonist of the formyl peptide receptor, a 2-fold increase in leukocyte emigration was noted in Fer(DR/DR) mice (p < 0.05). To determine whether these defects were due to Fer signaling in the endothelium or other nonhematopoietic cells, bone marrow chimeras were generated. WKYMVm-induced leukocyte recruitment in chimeric mice (WT bone marrow to Fer(DR/DR) recipients or vice versa) was similar to WT mice, suggesting that Fer kinase signaling in both leukocytes and endothelial cells serves to limit chemotaxis. Purified Fer(DR/DR) neutrophils demonstrated enhanced chemotaxis toward end target chemoattractants (WKYMVm and C5a) compared with WT using an under-agarose gel chemotaxis assay. These defects were not observed in response to intermediate chemoattractants (keratinocyte-derived cytokine, MIP-2, or LTB(4)). Increased WKYMVm-induced chemotaxis of Fer(DR/DR) neutrophils correlated with sustained PI3K activity and reduced reliance on the p38 MAPK pathway compared with WT neutrophils. Together, these data identify Fer as a novel inhibitory kinase for neutrophil chemotaxis toward end target chemoattractants through modulation of PI3K activity.

Endotyping in ARDS: one step forward in precision medicine.

BACKGROUND: The Berlin definition of acute respiratory distress syndrome (ARDS) includes only clinical characteristics. Understanding unique patient pathobiology may allow personalized treatment. We aimed to define and describe ARDS phenotypes/endotypes combining clinical and pathophysiologic parameters from a Canadian ARDS cohort. METHODS: A cohort of adult ARDS patients from multiple sites in Calgary, Canada, had plasma cytokine levels and clinical parameters measured in the first 24 h of ICU admission. We used a latent class model (LCM) to group the patients into several ARDS subgroups and identified the features differentiating those subgroups. We then discuss the subgroup effect on 30 day mortality. RESULTS: The LCM suggested three subgroups (n1 = 64, n2 = 86, and n3 = 30), and 23 out of 69 features made these subgroups distinct. The top five discriminating features were IL-8, IL-6, IL-10, TNF-a, and serum lactate. Mortality distinctively varied between subgroups. Individual clinical characteristics within the subgroup associated with mortality included mean PaO2/FiO2 ratio, pneumonia, platelet count, and bicarbonate negatively associated with mortality, while lactate, creatinine, shock, chronic kidney disease, vasopressor/ionotropic use, low GCS at admission, and sepsis were positively associated. IL-8 and Apache II were individual markers strongly associated with mortality (Area Under the Curve = 0.84). PERSPECTIVE: ARDS subgrouping using biomarkers and clinical characteristics is useful for categorizing a heterogeneous condition into several homogenous patient groups. This study found three ARDS subgroups using LCM; each subgroup has a different level of mortality. This model may also apply to developing further trial design, prognostication, and treatment selection.

Detection and Typing of Renal Amyloidosis by Fluorescence Spectroscopy Using the Environmentally Sensitive Fluorophore K114.

PURPOSE: To demonstrate that spectral analysis using the K114 fluorophore can detect and differentiate AL and AA renal amyloidosis. PROCEDURES: Kidney biopsies from patients with AL amyloidosis, AA amyloidosis, and normal samples with no evident pathology were stained with Congo Red and K114. The specimens were imaged on a spectral confocal microscope. RESULTS: Congo Red displayed homogeneous spectra across the three tissue types while K114 chromatically distinguished between normal tissue, AL amyloid, and AA amyloid. Additionally, Congo Red displayed an increased risk of false positive staining compared to K114. Spectral phasors computed from K114-stained tissue sections quantitatively differentiated the three tissue types. K114-stained amyloid deposits displayed a significantly greater increase in brightness after 50 images acquired in rapid succession compared to normal tissue. Quantitative analysis of intensity changes in the background of diseased tissue also differentiated AL and AA amyloid samples, suggesting widespread amyloid deposition. Both amyloid and the backgrounds of diseased samples red-shifted while normal tissue blue-shifted in response to repeated imaging, supporting this theory. CONCLUSIONS: K114 staining of renal biopsies is a promising technique to detect and differentiate types of renal amyloidosis. Due to the advantages this method has over traditional Congo Red staining, the techniques presented here warrant further development for potential use in clinical settings.

Sequential expression of IGF-IB followed by active TGF-ß1 induces synergistic pulmonary fibroproliferation in vivo.

Pulmonary fibrosis, the end stage of a variety of fibroproliferative lung diseases, is usually induced after repetitive or chronic lung injury or inflammation. The mechanisms of fibroproliferation are poorly understood. Insulin-like growth factor-I (IGF-I) is significantly elevated in patients with pulmonary fibrosis and fibroproliferative acute respiratory distress syndrome. However, we showed that IGF-I overexpression alone in wild-type mouse lungs does not cause fibroproliferation. We therefore questioned whether IGF-I, acting together with active TGF-ß1, a known profibrotic cytokine, enhances pulmonary fibroproliferation caused by active TGF-ß1. A unique sequential adenoviral transgene mouse model was used expressing AdEmpty/AdTGF-ß1 or AdhIGF-IB/AdTGF-ß1 transgenes. IGF-IB plus active TGF-ß1 transgene expression synergistically increased collagen deposition in the lung parenchyma compared with active TGF-ß1 expression alone. The enhanced fibrosis was accompanied by an increased recruitment of macrophages and lymphocytes into the bronchoalveolar lavage fluid (BALF) and inflammatory cells in the lungs. α-Smooth muscle actin expression, a marker of myofibroblast proliferation and differentiation, was also increased. Finally, fibroblasts exposed ex vivo to BALF isolated from AdhIGF-IB/AdTGF-ß1-transduced mice showed synergistic collagen induction compared with BALF from AdEmpty/AdTGF-ß1-transduced mice. This study provides the first direct evidence that IGF-I is able to synergistically enhance pulmonary fibroproliferation in cooperation with TGF-ß1.

A B1a-natural IgG-neutrophil axis is impaired in viral- and steroid-associated aspergillosis.

The lung naturally resists Aspergillus fumigatus (Af) in healthy individuals, but multiple conditions can disrupt this resistance, leading to lethal invasive infections. Core processes of natural resistance and its breakdown are undefined. We investigated three distinct conditions predisposing to lethal aspergillosis-severe SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection, influenza A viral pneumonia, and systemic corticosteroid use-in human patients and murine models. We found a conserved and essential coupling of innate B1a lymphocytes, Af-binding natural immunoglobulin G antibodies, and lung neutrophils. Failure of this axis concealed Af from neutrophils, allowing rapid fungal invasion and disease. Reconstituting the axis with immunoglobulin therapy reestablished resistance, thus representing a realistic pathway to repurpose currently available therapies. Together, we report a vital host resistance pathway that is responsible for protecting against life-threatening aspergillosis in the context of distinct susceptibilities.

Role of endothelial TLR4 for neutrophil recruitment into central nervous system microvessels in systemic inflammation.

Brain inflammation is a frequent consequence of sepsis and septic shock. We imaged leukocyte recruitment in brain postcapillary venules induced by i.p. administration of LPS as a simple model of systemic inflammation. The i.p. injection of LPS (0.5 mg/kg) induced significant leukocyte rolling and adhesion in brain postcapillary venules of wild-type (WT) mice and more than 90% were neutrophils. However, no emigrated neutrophils were detected in brain parenchyma. High levels of TNF-alpha and IL-1beta were detected in the plasma after LPS injection but a different profile (IL-1beta but not TNF-alpha) was detected in the brain. LPS caused no recruitment in TLR4 knockout mice. In chimeric mice with TLR4-expressing resident cells but TLR4-deficient bone marrow-derived circulating cells, neutrophil rolling and adhesion was similar to WT mice. This observation is consistent with a requirement for resident cells in the LPS-induced neutrophil recruitment into brain microvessels. Transgenic mice engineered to express TLR4 exclusively on endothelial cells had a similar level of leukocyte recruitment in brain as WT mice in response to LPS. High dose LPS (10 mg/kg) led to neutrophil infiltration in the brain parenchyma in WT mice. High KC and MIP-2 production was observed from brain parenchyma microglial cells, and CXCR2 knockout mice failed to recruit neutrophils. However, neither neutrophil infiltration nor KC or MIP-2 was observed in endothelial TLR4 transgenic mice in response to this LPS dose. Our results demonstrate that direct endothelial activation is sufficient to mediate leukocyte rolling and adhesion in cerebral microvessels but not sufficient for emigration to brain parenchyma.

Marginating transitional B cells modulate neutrophils in the lung during inflammation and pneumonia.

Pulmonary innate immunity is required for host defense; however, excessive neutrophil inflammation can cause life-threatening acute lung injury. B lymphocytes can be regulatory, yet little is known about peripheral transitional IgM+ B cells in terms of regulatory properties. Using single-cell RNA sequencing, we discovered eight IgM+ B cell subsets with unique gene regulatory networks in the lung circulation dominated by transitional type 1 B and type 2 B (T2B) cells. Lung intravital confocal microscopy revealed that T2B cells marginate in the pulmonary capillaries via CD49e and require CXCL13 and CXCR5. During lung inflammation, marginated T2B cells dampened excessive neutrophil vascular inflammation via the specialized proresolving molecule lipoxin A4 (LXA4). Exogenous CXCL13 dampened excessive neutrophilic inflammation by increasing marginated B cells, and LXA4 recapitulated neutrophil regulation in B cell-deficient mice during inflammation and fungal pneumonia. Thus, the lung microvasculature is enriched in multiple IgM+ B cell subsets with marginating capillary T2B cells that dampen neutrophil responses.

Adenovirus-mediated gene transfer of hIGF-IB in mouse lungs induced prolonged inflammation but no fibroproliferation.

Pulmonary fibrosis (PF), the end stage of a variety of fibroproliferative lung diseases, is characterized by excessive lung mesenchymal cell activation and extracellular matrix deposition. Most PF is induced after repetitive or chronic lung inflammation; however, a significant portion of PF occurs without apparent inflammation. The mechanisms of fibroproliferation are poorly understood. Studies have shown that cytokines regulating inflammation and tissue repair processes play essential roles in the development of PF. Insulin-like growth factor I (IGF-I) has been shown to stimulate lung mesenchymal cell proliferation and extracellular matrix synthesis in vitro and is significantly elevated in patients with PF. In this study, we investigated whether human IGF-IB (hIGF-IB) expression in the lungs induces PF in a C57BL/6 mouse model. Mice were subjected to adenoviral gene transfer, and the effects of hIGF-IB expression on the lungs were examined 3, 7, 14, 21, and 42 days after gene delivery. hIGF-IB expression induced significant and prolonged inflammatory cell infiltration into the lungs, with an early neutrophil infiltration followed by a late macrophage infiltration. No significant fibroblast or matrix accumulation could be detected in the lungs of these mice. No significant collagen accumulation could be detected in vivo, despite in vitro evidence that hIGF-IB induces collagen mRNA expression in fibroblasts. Therefore, IGF-IB alone is not sufficient to induce fibrosis, and it is possible that a coactivator is required to induce significant fibroproliferation in vivo.

Neuroimmune Responses Mediate Depression-Related Behaviors following Acute Colitis.

Many patients with visceral inflammation develop pain and psychiatric comorbidities such as major depressive disorder, worsening the quality of life and increasing the risk of suicide. Here we show that neuroimmune activation in mice with dextran sodium sulfate-induced colitis is accompanied by the development of pain and depressive behaviors. Importantly, treatment with the flavonoid luteolin prevented both neuroimmune responses and behavioral abnormalities, suggesting a new potential therapeutic approach for patients with inflammatory bowel diseases.

Targeting inflammatory monocytes in sepsis-associated encephalopathy and long-term cognitive impairment.

Sepsis-associated encephalopathy manifesting as delirium is a common problem in critical care medicine. In this study, patients that had delirium due to sepsis had significant cognitive impairments at 12-18 months after hospital discharge when compared with controls and Cambridge Neuropsychological Automated Test Battery-standardized scores in spatial recognition memory, pattern recognition memory, and delayed-matching-to-sample tests but not other cognitive functions. A mouse model of S. pneumoniae pneumonia-induced sepsis, which modeled numerous aspects of the human sepsis-associated multiorgan dysfunction, including encephalopathy, also revealed similar deficits in spatial memory but not new task learning. Both humans and mice had large increases in chemokines for myeloid cell recruitment. Intravital imaging of the brains of septic mice revealed increased neutrophil and CCR2+ inflammatory monocyte recruitment (the latter being far more robust), accompanied by subtle microglial activation. Prevention of CCR2+ inflammatory monocyte recruitment, but not neutrophil recruitment, reduced microglial activation and other signs of neuroinflammation and prevented all signs of cognitive impairment after infection. Therefore, therapeutically targeting CCR2+ inflammatory monocytes at the time of sepsis may provide a novel neuroprotective clinical intervention to prevent the development of persistent cognitive impairments.

Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs.

The rapid and selective accumulation of neutrophils into the lungs is thought to underlie the pulmonary failure that leads to sepsis-related death. In this study we investigated whether neutrophil TLR4 is important in LPS-induced pulmonary neutrophil recruitment by creating chimeric mice (transferring bone marrow between TLR4(+/+) and TLR4(-/-) mice). In TLR4(+/+) mice receiving TLR4(-/-) bone marrow, 6 weeks after transplant TLR4 was absent in all circulating leukocytes as well as in resident macrophages (these mice were termed LeukocyteTLR4(-/-)), and these cells were completely nonresponsive to LPS. In TLR4(-/-) mice receiving TLR4(+/+) bone marrow, endothelial cells but not leukocytes were deficient in TLR4 (EndotheliumTLR4(-/-)). Surprisingly, systemic LPS (0.5 mg/kg) induced a dramatic increase in neutrophil sequestration into the lungs of LeukocyteTLR4(-/-) mice over the first 4 hours. Concomitantly, numbers of circulating leukocytes decreased by 90%. By contrast, EndotheliumTLR4(-/-) mice showed very little increase in neutrophil sequestration in the lungs, suggesting that endothelium rather than leukocyte TLR4 was important. Intravital microscopy of peripheral microcirculation in the cremaster muscle revealed about 30-fold more leukocyte-endothelial cell interactions in LPS-treated EndotheliumTLR4(-/-) mice than in LPS-treated LeukocyteTLR4(-/-) mice. This is consistent with less sequestration of leukocytes into the lungs of EndotheliumTLR4(-/-) mice. In conclusion, our data challenge the view that LPS directly activates neutrophils to trap in lungs and suggest a far more important role than previously appreciated for the endothelial cells.

Enhanced production of IGF-I in the lungs of fibroproliferative ARDS patients.

Insulin-Like Growth Factor I (IGF-I) has been identified in the lungs of individuals with fibrotic lung diseases. In a previous retrospective study, we showed enhanced IGF-I immunoreactivity in individuals with fibroproliferative acute respiratory distress syndrome (FP-ARDS), but we were unable to determine if this correlation was causative. This study was undertaken to prospectively investigate whether IGF-I expression correlated with the fibroproliferative process and whether IGF-I was induced and made in the lungs. We measured IGF-I and procollagen III peptide (PCP-III) in the epithelial lining fluid (ELF) from controls, early ALI/ARDS patients and FP-ARDS patients. We also measured IGF-I mRNA and immunoreactivity from controls and FP-ARDS patient lung biopsies. We determined the level of lung permeability by measuring albumin and urea levels in ELF and serum. Our data show that IGF-I is significantly increased in the ELF in FP-ARDS patients. A significant correlation between IGF-I and PCP-III in the ELF of FP-ARDS patients is found. IGF-I mRNA is elevated in the FP-ARDS lung biopsies. Our data suggest that IGF-I found in the lungs of FP-ARDS patients results from both increased lung permeability and local production of IGF-I. The role of IGF-I in the fibroproliferative process in the lungs has recently been confirmed in an animal model of lung fibroproliferation. This study importantly suggest that IGF-I protein is made in the lungs of FP-ARDS patients and correlates with increased levels of ELF PCP-III, implicating a role for IGF-I in the fibroproliferative process in humans.

Characterization of S. pneumoniae pneumonia-induced multiple organ dysfunction syndrome: an experimental mouse model of gram-positive sepsis.

Streptococcus pneumoniae, a gram-positive bacteria, is the most common cause of community-acquired pneumonia. It is a common cause of septic shock with multiple organ dysfunction syndrome (MODS) resulting in significant mortality. Gram-positive mouse models of sepsis with MODS are required to examine mechanisms of immune responses in severe sepsis. To assess whether lung infection due to S. pneumoniae in a nonventilated mouse model can induce multiple organ dysfunction. S. pneumoniae, SPN 15814 strain, harvested at log phase, was injected intratracheally in C57BL/6 mice at OD 600 between 0.35 and 0.63. A dose of bacteria at OD 600 = 0.63 conferred approximately 30% mortality in 36 h. Lung pneumonia was assessed by histology, lung myeloperoxidase activity, and lung bacterial load; intestinal epithelial barrier integrity was assessed by measuring blood-to-lumen clearance of Cr-EDTA; renal function was assessed by measuring plasma creatinine and urea; and myocardiac function was assessed using an isolated perfused mouse heart model. S. pneumoniae-induced pneumonia resulted in neutrophil infiltration into the lungs and increased lung bacterial load. Although relatively few bacteria gained access to the blood stream, the pneumonia was accompanied by increased intestinal epithelial barrier permeability, increased plasma creatinine, and decreased cardiac output and stroke volume. These data clearly show that intratracheal S. pneumoniae induced not only pneumonia but also MODS, despite the fact that few organisms gain access to the blood stream. This model can be used as a good gram-positive model of sepsis and MODS for further studies.

Mice that exclusively express TLR4 on endothelial cells can efficiently clear a lethal systemic Gram-negative bacterial infection.

Recognition of LPS by TLR4 on immune sentinel cells such as macrophages is thought to be key to the recruitment of neutrophils to sites of infection with Gram-negative bacteria. To explore whether endothelial TLR4 plays a role in this process, we engineered and imaged mice that expressed TLR4 exclusively on endothelium (known herein as EndotheliumTLR4 mice). Local administration of LPS into tissue induced comparable neutrophil recruitment in EndotheliumTLR4 and wild-type mice. Following systemic LPS or intraperitoneal E. coli administration, most neutrophils were sequestered in the lungs of wild-type mice and did not accumulate at primary sites of infection. In contrast, EndotheliumTLR4 mice showed reduced pulmonary capillary neutrophil sequestration over the first 24 hours; as a result, they mobilized neutrophils to primary sites of infection, cleared bacteria, and resisted a dose of E. coli that killed 50% of wild-type mice in the first 48 hours. In fact, the only defect we detected in EndotheliumTLR4 mice was a failure to accumulate neutrophils in the lungs following intratracheal administration of LPS; this response required TLR4 on bone marrow-derived immune cells. Therefore, endothelial TLR4 functions as the primary intravascular sentinel system for detection of bacteria, whereas bone marrow-derived immune cells are critical for pathogen detection at barrier sites. Nonendothelial TLR4 contributes to failure to accumulate neutrophils at primary infection sites in a disseminated systemic infection.

Exogenous stromal cell-derived factor-1 induces modest leukocyte recruitment in vivo.

Stromal cell-derived factor-1 (SDF-1; CXCL12), a CXC chemokine, has been found to be involved in inflammation models in vivo and in cell adhesion, migration, and chemotaxis in vitro. This study aimed to determine whether exogenous SDF-1 induces leukocyte recruitment in mice. After systemic administration of SDF-1alpha, expression of the adhesion molecules P-selectin and VCAM-1 in mice was measured using a quantitative dual-radiolabeled Ab assay and leukocyte recruitment in various tissues was evaluated using intravital microscopy. The effect of local SDF-1alpha on leukocyte recruitment was also determined in cremaster muscle and compared with the effect of the cytokine TNFalpha and the CXC chemokine keratinocyte-derived chemokine (KC; CXCL1). Systemic administration of SDF-1alpha (10 microg, 4-5 h) induced upregulation of P-selectin, but not VCAM-1, in most tissues in mice. It caused modest leukocyte recruitment responses in microvasculature of cremaster muscle, intestine, and brain, i.e., an increase in flux of rolling leukocytes in cremaster muscle and intestines, leukocyte adhesion in all three tissues, and emigration in cremaster muscle. Local treatment with SDF-1alpha (1 microg, 4-5 h) reduced leukocyte rolling velocity and increased leukocyte adhesion and emigration in cremasteric venules, but the responses were much less profound than those elicited by KC or TNFalpha. SDF-1alpha-induced recruitment was dependent on endothelial P-selectin, but not P-selectin on platelets. We conclude that the exogenous SDF-1alpha enhances leukocyte-endothelial cell interactions and induces modest and endothelial P-selectin-dependent leukocyte recruitment.

Differential roles of CD36, ICAM-1, and P-selectin in Plasmodium falciparum cytoadherence in vivo.

Cytoadherence of Plasmodium falciparum-infected red blood cells (IRBCs) on human microvascular endothelium is mediated by synergistic adhesive interactions with different adhesion molecules in vitro. Here, the authors used a unique human/severe combined immunodeficient (SCID) mouse chimeric model to directly visualize IRBC-endothelial interactions in an intact human microvasculature in vivo. Stimulation of human skin grafts with 100 ng TNF-alpha for 4 h led to a dramatic reduction in the distance rolled by IRBCs before arrest, so that the majority of IRBCs adhered directly to the endothelium with a 1.8-fold increase in the number of adherent cells. The decrease in rolling distance and increase in adhesion could be reversed by anti-ICAM-1. More importantly, the effect of TNF-alpha could be seen only in the presence of CD36. A further increase in adhesion by 4.9-fold was observed after 24 h of TNF-alpha stimulation. The increase could be reversed by anti-ICAM-1, but not anti-VCAM-1. In histamine-stimulated grafts, the rolling flux fraction and adhesion increased by 2.8- and 1.6-fold, respectively. The increases were attributable to P-selectin as an inhibitory anti-P-selectin antibody abrogated both the increased rolling flux fraction and firm adhesion. These findings indicate that in addition to CD36, ICAM-1, and P-selectin are major contributors to the dynamic process of IRBC adhesion by different mechanisms in vivo.

Lipopolysaccharide: a p38 MAPK-dependent disrupter of neutrophil chemotaxis.

In sepsis, and in models of sepsis including endotoxemia, impaired neutrophil recruitment and chemotaxis have been reported. The inability of the endotoxemic neutrophil to chemotax could be attributed to the fact that intracellular signaling via LPS overrides signals from endogenous chemokines or, alternatively, that sequestration of neutrophils into lungs prevents access to peripheral tissues. Using both in vitro and in vivo chemotaxis assays the authors established that neutrophils from healthy mice chemotaxed in vivo toward MIP-2, whereas endotoxemic neutrophils did not. Since LPS activates leukocytes via the p38 MAPK pathway, SKF86002, a p38 MAPK inhibitor, was given to endotoxemic animals. SKF86002 significantly reversed the LPS-induced impairment in emigration of endotoxic neutrophils in response to MIP-2. Neutrophil chemotaxis in vitro was also impaired by LPS, via a p38 MAPK-dependent pathway, and this impairment could be reversed via p38 MAPK inhibition. Although neutrophil numbers dropped in the circulation and trapped in lungs during endotoxemia, SKF86002 did not reverse these parameters, demonstrating that p38 MAPK inhibition did not release trapped neutrophils from the lungs. In conclusion, the data suggest that the impaired emigration and chemotaxis of neutrophils at peripheral sites during endotoxemia may be partially due to a p38 MAPK-mediated inhibition of neutrophil responses to endogenous chemokines.

Platelets express functional Toll-like receptor-4.

Profound thrombocytopenia occurs in humans with sepsis and in mice administered lipopolysaccharide (LPS). Growing evidence indicates that platelets may contribute to these abnormalities, but whether that is a direct result of LPS activation of platelets or an indirect result of other inflammatory mechanisms remains unclear. Here we demonstrate that although platelets do not increase P-selectin expression in response to LPS, platelets bind more avidly to fibrinogen under flow conditions in a Toll-like receptor-4 (TLR4)-dependent manner. In addition, we find that CD41+ megakaryocytes grown from fetal livers and adult circulating platelets express significant amounts of TLR4. LPS induced thrombocytopenia in wild-type mice but not in TLR4-deficient (TLR4def) mice. Wild-type platelets accumulated in the lungs of wild-type mice in response to LPS; TLR4def platelets did not. However, wild-type platelets did not accumulate in the lungs of LPS-treated TLR4def mice. Neutrophils also accumulated in the lungs, and this preceded platelet accumulation. Neutrophil depletion completely abolished LPS-induced platelet sequestration into the lungs, but platelet depletion did not affect neutrophil accumulation. Thus, our data show for the first time that platelets do express functional levels of TLR4, which contribute to thrombocytopenia through neutrophil-dependent pulmonary sequestration in response to LPS.