Neutrophils Regulate Tissue Neutrophilia in Inflammation via the Oxidant-modified Lipid Lysophosphatidylserine

Resolution of neutrophilia characteristic of acute inflammation requires cessation of neutrophil recruitment and removal of tissue neutrophils. Based on in vitro studies, a role in these events was hypothesized for oxidant-generated lysophosphatidylserine (lyso-PS) on recruited neutrophils signaling via the G2A receptor on macrophages. Peritoneal exudate neutrophils harvested from wild type (WT) mice had 5-fold more lyso-PS (lyso-PS^high) than those of gp91^phox−/− (lyso-PS^low) mice. Ex vivo engulfment of lyso-PS^high neutrophils (95% viable) by WT peritoneal macrophages was quantitatively similar to UV-irradiated apoptotic blood neutrophils, although the signaling pathway for the former was uniquely dependent on macrophage G2A. In contrast, lyso-PS^low neutrophils were poorly engulfed unless presented with exogenous lyso-PS. Enhanced clearance of lyso-PS^high neutrophils was also seen in vivo following their adoptive transfer into inflamed peritonea of WT but not G2A^−/− mice, further supporting a requirement for signaling via G2A. To investigate downstream effects of lyso-PS/G2A signaling, antibody blockade of G2A in WT mice reduced macrophage CD206 expression and efferocytosis during peritonitis. Conversely, adoptive transfer of lyso-PS^high neutrophils early in inflammation in gp91^phox−/− mice led to accelerated development of efferocytic^high and CD206^high macrophages. This macrophage reprogramming was associated with suppressed production of pro-inflammatory mediators and reduced neutrophilia. These effects were not seen if G2A was blocked or lyso-PS^low neutrophils were transferred. Taken together, the results demonstrate that oxidant-generated lyso-PS made by viable tissue neutrophils is an endogenous anti-inflammatory mediator working in vivo to orchestrate the “early” and rapid clearance of recruited neutrophils as well as the reprogramming of “resolving” macrophages.     

Macrophage SR-BI mediates efferocytosis via Src/PI3K/Rac1 signaling and reduces atherosclerotic lesion necrosis

Macrophage apoptosis and efferocytosis are key determinants of atherosclerotic plaque inflammation and necrosis. Bone marrow transplantation studies in ApoE- and LDLR-deficient mice revealed that hematopoietic scavenger receptor class B type I (SR-BI) deficiency results in severely defective efferocytosis in mouse atherosclerotic lesions, resulting in a 17-fold higher ratio of free to macrophage-associated dead cells in lesions containing SR-BI^−/− cells, 5-fold more necrosis, 65.2% less lesional collagen content, nearly 7-fold higher dead cell accumulation, and 2-fold larger lesion area. Hematopoietic SR-BI deletion elicited a maladaptive inflammatory response [higher interleukin (IL)-1β, IL-6, and TNF-α lower IL-10 and transforming growth factor β]. Efferocytosis of apoptotic thymocytes was reduced by 64% in SR-BI^−/− versus WT macrophages, both in vitro and in vivo. In response to apoptotic cells, macrophage SR-BI bound with phosphatidylserine and induced Src phosphorylation and cell membrane recruitment, which led to downstream activation of phosphoinositide 3-kinase (PI3K) and Ras-related C3 botulinum toxin substrate 1 (Rac1) for engulfment and clearance of apoptotic cells, as inhibition of Src decreased PI3K, Rac1-GTP, and efferocytosis in WT cells. Pharmacological inhibition of Rac1 reduced macrophage efferocytosis in a SR-BI-dependent fashion, and activation of Rac1 corrected the defective efferocytosis in SR-BI^−/− macrophages. Thus, deficiency of macrophage SR-BI promotes defective efferocytosis signaling via the Src/PI3K/Rac1 pathway, resulting in increased plaque size, necrosis, and inflammation.     

Galectin-3 Ablation Enhances Liver Steatosis,but Attenuates Inflammation and IL-33-Dependent Fibrosis in Obesogenic Mouse Model of Nonalcoholic Steatohepatitis

The importance of Galectin-3 (Gal-3) in obesity-associated liver pathology is incompletely defined. To dissect the role of Gal-3 in fibrotic nonalcoholic steatohepatitis (NASH), Gal-3-deficient (LGALS3^−/−) and wild-type (LGALS3^+/+) C57Bl/6 mice were placed on an obesogenic high fat diet (HFD, 60% kcal fat) or standard chow diet for 12 and 24 wks. Compared to WT mice, HFD-fed LGALS3^−/− mice developed, in addition to increased visceral adiposity and diabetes, marked liver steatosis, which was accompanied with higher expression of hepatic PPAR-γ, Cd36, Abca-1 and FAS. However, as opposed to LGALS3^−/− mice, hepatocellular damage, inflammation and fibrosis were more extensive in WT mice which had an elevated number of mature myeloid dendritic cells, proinflammatory CD11b⁺Ly6C^hi monocytes/macrophages in liver, peripheral blood and bone marrow, and increased hepatic CCL2, F4/80, CD11c, TLR4, CD14, NLRP3 inflammasome, IL-1β and NADPH-oxidase enzymes mRNA expression. Thus, obesity-driven greater steatosis was uncoupled with attenuated fibrotic NASH in Gal-3-deficient mice. HFD-fed WT mice had a higher number of hepatocytes that strongly expressed IL-33 and hepatic CD11b⁺IL-13⁺ cells, increased levels of IL-33 and IL-13 and up-regulated IL-33, ST2 and IL-13 mRNA in liver compared with LGALS3^−/− mice. IL-33 failed to induce ST2 upregulation and IL-13 production by LGALS3^−/− peritoneal macrophages in vitro. Administration of IL-33 in vivo enhanced liver fibrosis in HFD-fed mice in both genotypes, albeit to a significantly lower extent in LGALS3^−/− mice, which was associated with less numerous hepatic IL-13-expressing CD11b⁺ cells. The present study provides evidence of a novel role for Gal-3 in regulating IL-33-dependent liver fibrosis.     

High mobility group protein-1 inhibits phagocytosis of apoptotic neutrophils through binding to phosphatidylserine

Phagocytosis of apoptotic cells, also called efferocytosis, is an essential feature of immune responses and critical to resolution of inflammation. Impaired efferocytosis is associated with an unfavorable outcome from inflammatory diseases, including acute lung injury and pulmonary manifestations of cystic fibrosis. High mobility group protein-1 (HMGB1), a nuclear nonhistone DNA-binding protein, has recently been found to be secreted by immune cells upon stimulation with LPS and cytokines. Plasma and tissue levels of HMGB1 are elevated for prolonged periods in chronic and acute inflammatory conditions, including sepsis, rheumatoid arthritis, acute lung injury, burns, and hemorrhage. In this study, we found that HMGB1 inhibits phagocytosis of apoptotic neutrophils by macrophages in vivo and in vitro. Phosphatidylserine (PS) is directly involved in the inhibition of phagocytosis by HMGB1, as blockade of HMGB1 by PS eliminates the effects of HMGB1 on efferocytosis. Confocal and fluorescence resonance energy transfer demonstrate that HMGB1 interacts with PS on the neutrophil surface. However, HMGB1 does not inhibit PS-independent phagocytosis of viable neutrophils. Bronchoalveolar lavage fluid from Scnn(+) mice, a murine model of cystic fibrosis lung disease which contains elevated concentrations of HMGB1, inhibits neutrophil efferocytosis. Anti-HMGB1 Abs reverse the inhibitory effect of Scnn(+) bronchoalveolar lavage on efferocytosis, showing that this effect is due to HMGB1. These findings demonstrate that HMGB1 can modulate phagocytosis of apoptotic neutrophils and suggest an alternative mechanism by which HMGB1 is involved in enhancing inflammatory responses.     

IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

Interleukin (IL-) 36 cytokines (previously designated as novel IL-1 family member cytokines; IL-1F5– IL-1F10) constitute a novel cluster of cytokines structurally and functionally similar to members of the IL-1 cytokine cluster. The effects of IL-36 cytokines in inflammatory lung disorders remains poorly understood. The current study sought to investigate the effects of IL-36α (IL-1F6) and test the hypothesis that IL-36α acts as a pro-inflammatory cytokine in the lung in vivo. Intratracheal instillation of recombinant mouse IL-36α induced neutrophil influx in the lungs of wild-type C57BL/6 mice and IL-1αβ^−/− mice in vivo. IL-36α induced neutrophil influx was also associated with increased mRNA expression of neutrophil-specific chemokines CXCL1 and CXCL2 in the lungs of C57BL/6 and IL-1αβ^−/− mice in vivo. In addition, intratracheal instillation of IL-36α enhanced mRNA expression of its receptor IL-36R in the lungs of C57BL/6 as well as IL-1αβ^−/− mice in vivo. Furthermore, in vitro incubation of CD11c⁺ cells with IL-36α resulted in the generation of neutrophil-specific chemokines CXCL1, CXCL2 as well as TNFα. IL-36α increased the expression of the co-stimulatory molecule CD40 and enhanced the ability of CD11c⁺ cells to induce CD4⁺ T cell proliferation in vitro. Furthermore, stimulation with IL-36α activated NF-κB in a mouse macrophage cell line. These results demonstrate that IL-36α acts as a pro-inflammatory cytokine in the lung without the contribution of IL-1α and IL-1β. The current study describes the pro-inflammatory effects of IL-36α in the lung, demonstrates the functional redundancy of IL-36α with other agonist cytokines in the IL-1 and IL-36 cytokine cluster, and suggests that therapeutic targeting of IL-36 cytokines could be beneficial in inflammatory lung diseases.     

γδ T Cells Are Required for Pulmonary IL-17A Expression after Ozone Exposure in Mice: Role of TNFα

Ozone is an air pollutant that causes pulmonary symptoms. In mice, ozone exposure causes pulmonary injury and increases bronchoalveolar lavage macrophages and neutrophils. We have shown that IL-17A is important in the recruitment of neutrophils after subacute ozone exposure (0.3 ppm for 24–72 h). We hypothesized that γδ T cells are the main producers of IL-17A after subacute ozone. To explore this hypothesis we exposed wildtype mice and mice deficient in γδ T cells (TCRδ^−/−) to ozone or room air. Ozone-induced increases in BAL macrophages and neutrophils were attenuated in TCRδ^−/− mice. Ozone increased the number of γδ T cells in the lungs and increased pulmonary Il17a mRNA expression and the number of IL-17A⁺ CD45⁺ cells in the lungs and these effects were abolished in TCRδ^−/− mice. Ozone-induced increases in factors downstream of IL-17A signaling, including G-CSF, IL-6, IP-10 and KC were also decreased in TCRδ^−/− versus wildtype mice. Neutralization of IL-17A during ozone exposure in wildtype mice mimicked the effects of γδ T cell deficiency. TNFR2 deficiency and etanercept, a TNFα antagonist, also reduced ozone-induced increases in Il17a mRNA, IL-17A⁺ CD45⁺ cells and BAL G-CSF as well as BAL neutrophils. TNFR2 deficient mice also had decreased ozone-induced increases in Ccl20, a chemoattractant for IL-17A⁺ γδ T cells. Il17a mRNA and IL-17A⁺ γδ T cells were also lower in obese Cpe^fat versus lean WT mice exposed to subacute ozone, consistent with the reduced neutrophil recruitment observed in the obese mice. Taken together, our data indicate that pulmonary inflammation induced by subacute ozone requires γδ T cells and TNFα-dependent recruitment of IL-17A⁺ γδ T cells to the lung.     

Role of IL-1β in Experimental Cystic Fibrosis upon P. aeruginosa Infection

Cystic fibrosis is associated with increased inflammatory responses to pathogen challenge. Here we revisited the role of IL-1β in lung pathology using the experimental F508del-CFTR murine model on C57BL/6 genetic background (Cftr ^tm1eur or d/d), on double deficient for d/d and type 1 interleukin-1 receptor (d/d X IL-1R1^−/−), and antibody neutralization. At steady state, young adult d/d mice did not show any signs of spontaneous lung inflammation. However, IL-1R1 deficiency conferred partial protection to repeated P. aeruginosa endotoxins/LPS lung instillation in d/d mice, as 50% of d/d mice succumbed to inflammation, whereas all d/d x IL-1R1^−/− double mutants survived with lower initial weight loss and less pulmonary collagen and mucus production, suggesting that the absence of IL-1R1 signaling is protective in d/d mice in LPS-induced lung damage. Using P. aeruginosa acute lung infection we found heightened neutrophil recruitment in d/d mice with higher epithelial damage, increased bacterial load in BALF, and augmented IL-1β and TNF-α in parenchyma as compared to WT mice. Thus, F508del-CFTR mice show enhanced IL-1β signaling in response to P. aeruginosa. IL-1β antibody neutralization had no effect on lung homeostasis in either d/d or WT mice, however P. aeruginosa induced lung inflammation and bacterial load were diminished by IL-1β antibody neutralization. In conclusion, enhanced susceptibility to P. aeruginosa in d/d mice correlates with an excessive inflammation and with increased IL-1β production and reduced bacterial clearance. Further, we show that neutralization of IL-1β in d/d mice through the double mutation d/d x IL-1R1^−/− and in WT via antibody neutralization attenuates inflammation. This supports the notion that intervention in the IL-1R1/IL-1β pathway may be detrimental in CF patients.     

The Role of Toll-Like Receptor 2 in Inflammation and Fibrosis during Progressive Renal Injury

Tissue fibrosis and chronic inflammation are common causes of progressive organ damage, including progressive renal disease, leading to loss of physiological functions. Recently, it was shown that Toll-like receptor 2 (TLR2) is expressed in the kidney and activated by endogenous danger signals. The expression and function of TLR2 during renal fibrosis and chronic inflammation has however not yet been elucidated. Therefore, we studied TLR2 expression in human and murine progressive renal diseases and explored its role by inducing obstructive nephropathy in TLR2^−/− or TLR2^+/+ mice. We found that TLR2 is markedly upregulated on tubular and tubulointerstitial cells in patients with chronic renal injury. In mice with obstructive nephropathy, renal injury was associated with a marked upregulation and change in distribution of TLR2 and upregulation of murine TLR2 danger ligands Gp96, biglycan, and HMGB1. Notably, TLR2 enhanced inflammation as reflected by a significantly reduced influx of neutrophils and production of chemokines and TGF-β in kidneys of TLR2^−/− mice compared with TLR2^+/+ animals. Although, the obstructed kidneys of TLR2^−/− mice had less interstitial myofibroblasts in the later phase of obstructive nephropathy, tubular injury and renal matrix accumulation was similar in both mouse strains. Together, these data demonstrate that TLR2 can initiate renal inflammation during progressive renal injury and that the absence of TLR2 does not affect the development of chronic renal injury and fibrosis.     

NLRP3 Protein Deficiency Exacerbates Hyperoxia-induced Lethality through Stat3 Protein Signaling Independent of Interleukin-1β

Supplemental oxygen inhalation is frequently used to treat severe respiratory failure; however, prolonged exposure to hyperoxia causes hyperoxic acute lung injury (HALI), which induces acute respiratory distress syndrome and leads to high mortality rates. Recent investigations suggest the possible role of NLRP3 inflammasomes, which regulate IL-1β production and lead to inflammatory responses, in the pathophysiology of HALI; however, their role is not fully understood. In this study, we investigated the role of NLRP3 inflammasomes in mice with HALI. Under hyperoxic conditions, NLRP3^−/− mice died at a higher rate compared with wild-type and IL-1β^−/− mice, and there was no difference in IL-1β production in their lungs. Under hyperoxic conditions, the lungs of NLRP3^−/− mice exhibited reduced inflammatory responses, such as inflammatory cell infiltration and cytokine expression, as well as increased and decreased expression of MMP-9 and Bcl-2, respectively. NLRP3^−/− mice exhibited diminished expression and activation of Stat3, which regulates MMP-9 and Bcl-2, in addition to increased numbers of apoptotic alveolar epithelial cells. In vitro experiments revealed that alveolar macrophages and neutrophils promoted Stat3 activation in alveolar epithelial cells. Furthermore, NLRP3 deficiency impaired the migration of neutrophils and chemokine expression by macrophages. These findings demonstrate that NLRP3 regulates Stat3 signaling in alveolar epithelial cells by affecting macrophage and neutrophil function independent of IL-1β production and contributes to the pathophysiology of HALI.     

The role of pro- and anti-inflammatory responses in silica-induced lung fibrosis

Background

It has been generally well accepted that chronic inflammation is a necessary component of lung fibrosis but this concept has recently been challenged.

Methods

Using biochemical, histological, immunohistochemistry, and cellular analyses, we compared the lung responses (inflammation and fibrosis) to fibrogenic silica particles (2.5 and 25 mg/g lung) in Sprague-Dawley rats and NMRI mice.

Results

Rats treated with silica particles developed chronic and progressive inflammation accompanied by an overproduction of TNF-α as well as an intense lung fibrosis. Dexamethasone or pioglitazone limited the amplitude of the lung fibrotic reaction to silica in rats, supporting the paradigm that inflammation drives lung fibrosis.In striking contrast, in mice, silica induced only a limited and transient inflammation without TNF-α overproduction. However, mice developed lung fibrosis of a similar intensity than rats. The fibrotic response in mice was accompanied by a high expression of the anti-inflammatory and fibrotic cytokine IL-10 by silica-activated lung macrophages. In mice, IL-10 was induced only by fibrotic particles and significantly expressed in the lung of silica-sensitive but not silica-resistant strains of mice. Anti-inflammatory treatments did not control lung fibrosis in mice.

Conclusion

These results indicate that, beside chronic lung inflammation, a pronounced anti-inflammatory reaction may also contribute to the extension of silica-induced lung fibrosis and represents an alternative pathway leading to lung fibrosis.     

Decay-Accelerating Factor 1 Deficiency Exacerbates Leptospiral-Induced Murine Chronic Nephritis and Renal Fibrosis

Leptospirosis is a global zoonosis caused by pathogenic Leptospira, which can colonize the proximal renal tubules and persist for long periods in the kidneys of infected hosts. Here, we characterized the infection of C57BL/6J wild-type and Daf1^−/− mice, which have an enhanced host response, with a virulent Leptospira interrogans strain at 14 days post-infection, its persistence in the kidney, and its link to kidney fibrosis at 90 days post-infection. We found that Leptospira interrogans can induce acute moderate nephritis in wild-type mice and is able to persist in some animals, inducing fibrosis in the absence of mortality. In contrast, Daf1^−/− mice showed acute mortality, with a higher bacterial burden. At the chronic stage, Daf1^−/− mice showed greater inflammation and fibrosis than at 14 days post-infection and higher levels at all times than the wild-type counterpart. Compared with uninfected mice, infected wild-type mice showed higher levels of IL-4, IL-10 and IL-13, with similar levels of α-smooth muscle actin, galectin-3, TGF-β1, IL-17, IFN-γ, and lower IL-12 levels at 90 days post-infection. In contrast, fibrosis in Daf1^−/− mice was accompanied by high expression of α-smooth muscle actin, galectin-3, IL-10, IL-13, and IFN-γ, similar levels of TGF-β1, IL-12, and IL-17 and lower IL-4 levels. This study demonstrates the link between Leptospira-induced murine chronic nephritis with renal fibrosis and shows a protective role of Daf1.     

IL-17A Modulates Oxidant Stress-Induced Airway Hyperresponsiveness but Not Emphysema

IL-17A induces the release of pro-inflammatory cytokines and of reactive oxygen species which could lead to neutrophilic inflammation. We determined the role of IL-17 receptor (IL-17R) signalling in oxidant-induced lung emphysema and airway hyperresponsiveness. IL-17R^−/− and wild-type C57/BL6 mice were exposed to ozone (3 ppm; 3 hours) for 12 times over 6 weeks. Bronchial responsiveness to acetylcholine was measured, and lungs were retrieved. Mean linear intercept (L_m) and isometric contractile responses of intrapulmonary airways to acetylcholine were determined. In wild-type mice but not in IL-17R^−/−, chronic ozone exposure caused airway hyperresponsiveness. The increase in L_m after chronic ozone exposure of wild-type mice was also observed in IL-17R^−/− mice. The increased maximal contractile response to acetylcholine seen in airways of wild-type mice exposed to ozone was abolished in IL-17R^−/− mice. p38-mitogen-activated protein kinase (MAPK) and dexamethasone-dependent increase in contractile response was reduced in airways from IL-17R^−/− ozone-exposed mice. Lung inflammation scores were not altered in IL-17R^−/− mice exposed to ozone compared to wild-type mice. The increased release of IL-17 and IL-1β, and the activation of p38 MAPK in the lungs of ozone-exposed mice was reduced in IL-17R^−/− mice. IL-17R signalling underlies the increase in airway hyperresponsiveness seen after ozone exposure, mediated by the increased contractility of airway smooth muscle. The emphysema and lung inflammation induced by ozone is not dependent on IL-17.     

Genetic ablation of caveolin-2 sensitizes mice to bleomycin-induced injury

Caveolar domains act as platforms for the organization of molecular complexes involved in signal transduction. Caveolin proteins, the principal structural components of caveolae, have been involved in many cellular processes. Caveolin-1 (Cav-1) and caveolin-2 (Cav-2) are highly expressed in the lung. Cav-1-deficient mice (Cav-1^−/−) and Cav-2-deficient mice (Cav-2^−/−) exhibit severe lung dysfunction attributed to a lack of Cav-2 expression. Recently, Cav-1 has been shown to regulate lung fibrosis in different models. Here, we show that Cav-2 is also involved in modulation of the fibrotic response, but through distinct mechanisms. Treatment of wild-type mice with the pulmonary fibrosis-inducer bleomycin reduced the expression of Cav-2 and its phosphorylation at tyrosine 19. Importantly, Cav-2^−/− mice, but not Cav-1^−/− mice, were more sensitive to bleomycin-induced lung injury in comparison to wild-type mice. Bleomycin-induced lung injury was characterized by alveolar thickening, increase in cell density, and extracellular matrix deposition. The lung injury observed in bleomycin-treated Cav-2^−/− mice was not associated with alterations in the TGF-β signaling pathway and/or in the ability to produce collagen. However, apoptosis and proliferation were more prominent in lungs of bleomycin-treated Cav-2^−/− mice. Since Cav-1^−/− mice also lack Cav-2 expression and show a different outcome after bleomycin treatment, we conclude that Cav-1 and Cav-2 have distinct roles in bleomycin induced-lung fibrosis, and that the balance of both proteins determines the development of the fibrotic process.     

Protective Role of P2Y2 Receptor against Lung Infection Induced by Pneumonia Virus of Mice

ATP released in the early inflammatory processes acts as a danger signal by binding to purinergic receptors expressed on immune cells. A major contribution of the P2Y₂ receptor of ATP/UTP to dendritic cell function and Th2 lymphocyte recruitment during asthmatic airway inflammation was previously reported. We investigated here the involvement of P2Y₂ receptor in lung inflammation initiated by pneumonia virus of mice infection. We demonstrated that P2Y₂ ^−/− mice display a severe increase in morbidity and mortality rate in response to the virus. Lower survival of P2Y₂ ^−/− mice was not significantly correlated with excessive inflammation despite the higher level of neutrophil recruiters in their bronchoalveolar fluids. Interestingly, we observed reduced ATP level and lower numbers of dendritic cells, CD4⁺ T cells and CD8⁺ T cells in P2Y₂ ^−/− compared to P2Y₂ ^+/+ infected lungs. Lower level of IL-12 and higher level of IL-6 in bronchoalveolar fluid support an inhibition of Th1 response in P2Y₂ ^−/− infected mice. Quantification of DC recruiter expression revealed comparable IP-10 and MIP-3α levels but a reduced BRAK level in P2Y₂ ^−/− compared to P2Y₂ ^+/+ bronchoalveolar fluids. The increased morbidity and mortality of P2Y₂ ^−/− mice could be the consequence of a lower viral clearance leading to a more persistent viral load correlated with the observed higher viral titer. The decreased viral clearance could result from the defective Th1 response to PVM with a lack of DC and T cell infiltration. In conclusion, P2Y₂ receptor, previously described as a target in cystic fibrosis therapy and as a mediator of Th2 response in asthma, may also regulate Th1 response protecting mice against lung viral infection.     

Thy-1 Attenuates TNF-α-Activated Gene Expression in Mouse Embryonic Fibroblasts via Src Family Kinase

Heterogeneous surface expression of Thy-1 in fibroblasts modulates inflammation and may thereby modulate injury and repair. As a paradigm, patients with idiopathic pulmonary fibrosis, a disease with pathologic features of chronic inflammation, demonstrate an absence of Thy-1 immunoreactivity within areas of fibrotic activity (fibroblast foci) in contrast to the predominant Thy-1 expressing fibroblasts in the normal lung. Likewise, Thy-1 deficient mice display more severe lung fibrosis in response to an inflammatory injury than wildtype littermates. We investigated the role of Thy-1 in the response of fibroblasts to the pro-inflammatory cytokine TNF-α. Our study demonstrates distinct profiles of TNF-α-activated gene expression in Thy-1 positive (Thy-1+) and negative (Thy-1−) subsets of mouse embryonic fibroblasts (MEF). TNF-α induced a robust activation of MMP-9, ICAM-1, and the IL-8 promoter driven reporter in Thy-1− MEFs, in contrast to only a modest increase in Thy-1+ counterparts. Consistently, ectopic expression of Thy-1 in Thy-1− MEFs significantly attenuated TNF-α-activated gene expression. Mechanistically, TNF-α activated Src family kinase (SFK) only in Thy-1− MEFs. Blockade of SFK activation abrogated TNF-α-activated gene expression in Thy-1− MEFs, whereas restoration of SFK activation rescued the TNF-α response in Thy-1+ MEFs. Our findings suggest that Thy-1 down-regulates TNF-α-activated gene expression via interfering with SFK- and NF-κB-mediated transactivation. The current study provides a novel mechanistic insight to the distinct roles of fibroblast Thy-1 subsets in inflammation.     

CD36 and Fyn Kinase Mediate Malaria-Induced Lung Endothelial Barrier Dysfunction in Mice Infected with Plasmodium berghei

Severe malaria can trigger acute lung injury characterized by pulmonary edema resulting from increased endothelial permeability. However, the mechanism through which lung fluid conductance is altered during malaria remains unclear. To define the role that the scavenger receptor CD36 may play in mediating this response, C57BL/6J (WT) and CD36−/− mice were infected with P. berghei ANKA and monitored for changes in pulmonary endothelial barrier function employing an isolated perfused lung system. WT lungs demonstrated a >10-fold increase in two measures of paracellular fluid conductance and a decrease in the albumin reflection coefficient (σ_alb) compared to control lungs indicating a loss of barrier function. In contrast, malaria-infected CD36−/− mice had near normal fluid conductance but a similar reduction in σ_alb. In WT mice, lung sequestered iRBCs demonstrated production of reactive oxygen species (ROS). To determine whether knockout of CD36 could protect against ROS-induced endothelial barrier dysfunction, mouse lung microvascular endothelial monolayers (MLMVEC) from WT and CD36−/− mice were exposed to H₂O₂. Unlike WT monolayers, which showed dose-dependent decreases in transendothelial electrical resistance (TER) from H₂O₂ indicating loss of barrier function, CD36−/− MLMVEC demonstrated dose-dependent increases in TER. The differences between responses in WT and CD36−/− endothelial cells correlated with important differences in the intracellular compartmentalization of the CD36-associated Fyn kinase. Malaria infection increased total lung Fyn levels in CD36−/− lungs compared to WT, but this increase was due to elevated production of the inactive form of Fyn further suggesting a dysregulation of Fyn-mediated signaling. The importance of Fyn in CD36-dependent endothelial signaling was confirmed using in vitro Fyn knockdown as well as Fyn−/− mice, which were also protected from H₂O₂- and malaria-induced lung endothelial leak, respectively. Our results demonstrate that CD36 and Fyn kinase are critical mediators of the increased lung endothelial fluid conductance caused by malaria infection.     

Neutrophils Promote Mycobacterial Trehalose Dimycolate-Induced Lung Inflammation via the Mincle Pathway

Trehalose 6,6′-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFα production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincle^−/− mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses.     

Absence of the Common Gamma Chain (γc), a Critical Component of the Type I IL-4 Receptor,Increases the Severity of Allergic Lung Inflammation

The T_H2 cytokines, IL-4 and IL-13, play critical roles in inducing allergic lung inflammation and drive the alternative activation of macrophages (AAM). Although both cytokines share receptor subunits, IL-4 and IL-13 have differential roles in asthma pathogenesis: IL-4 regulates T_H2 cell differentiation, while IL-13 regulates airway hyperreactivity and mucus production. Aside from controlling T_H2 differentiation, the unique contribution of IL-4 signaling via the Type I receptor in airway inflammation remains unclear. Therefore, we analyzed responses in mice deficient in gamma c (γ_c) to elucidate the role of the Type I IL-4 receptor. OVA primed CD4⁺ OT-II T cells were adoptively transferred into RAG2^−/− and γ_c ^−/− mice and allergic lung disease was induced. Both γ_c ^−/− and γ_cxRAG2^−/− mice developed increased pulmonary inflammation and eosinophilia upon OVA challenge, compared to RAG2^−/− mice. Characteristic AAM proteins FIZZ1 and YM1 were expressed in lung epithelial cells in both mouse strains, but greater numbers of FIZZ1+ or YM1+ airways were present in γ_c ^−/− mice. Absence of γ_c in macrophages, however, resulted in reduced YM1 expression. We observed higher T_H2 cytokine levels in the BAL and an altered DC phenotype in the γ_c ^−/− recipient mice suggesting the potential for dysregulated T cell and dendritic cell (DC) activation in the γ_c-deficient environment. These results demonstrate that in absence of the Type I IL-4R, the Type II R can mediate allergic responses in the presence of T_H2 effectors. However, the Type I R regulates AAM protein expression in macrophages.     

IL-17, produced by lymphocytes and neutrophils,is necessary for lipopolysaccharide-induced airway neutrophilia: IL-15 as a possible trigger

IL-17 is a cytokine implicated in the regulation of inflammation. We investigated the role of this cytokine in neutrophil recruitment using a model of LPS-induced lung inflammation in mice. In the bronchoalveolar lavage, LPS induced a first influx of neutrophils peaking at day 1, followed by a second wave, peaking at day 2. IL-17 levels were increased during the late phase neutrophilia (day 2), and this was concomitant with an increased number of T cells and macrophages, together with an increase of KC and macrophage-inflammatory protein-2 levels in the lung tissue. Intranasal treatment with a neutralizing murine anti-IL-17 Ab inhibited the late phase neutrophilia. In the bronchoalveolar lavage cells, IL-17 mRNA was detected at days 1, 2, and 3 postchallenge, with a strong expression at day 2. This expression was associated with CD4(+) and CD8(+) cells, but also with neutrophils. When challenged with LPS, despite the absence of T cells, SCID mice also developed a neutrophilic response associated with IL-17 production. In BALB/c mice, IL-15 mRNA, associated mainly with neutrophils, was evidenced 1 day after LPS challenge. In vitro, IL-15 was able to induce IL-17 release from purified spleen CD4(+) cells, but not spleen CD8(+) or airway neutrophils. We have shown that IL-17, produced mainly by CD4(+) cells, but also by neutrophils, plays a role in the mobilization of lung neutrophils following bacterial challenge. In addition, our results suggest that IL-15 could represent a physiological trigger that leads to IL-17 production following bacterial infection.