Inflammatory Mononuclear Phagocytes Research Articles

Interferon Lambda Regulates Cellular and Humoral Immunity in Pristane-Induced Lupus

A pivotal role of type I interferons in systemic lupus erythematosus (SLE) is widely accepted. Type III interferons (IFN-λ) however, the most recently discovered cytokines grouped within the interferon family, have not been extensively studied in lupus disease models yet. Growing evidence suggests a role for IFN-λ in regulating both innate and adaptive immune responses, and increased serum concentrations have been described in multiple autoimmune diseases including SLE. Using the pristane-induced lupus model, we found that mice with defective IFN-λ receptors (Ifnlr1−/−) showed increased survival rates, decreased lipogranuloma formation and reduced anti-dsDNA autoantibody titers in the early phase of autoimmunity development compared to pristane-treated wild-type mice. Moreover, Ifnlr1−/− mice treated with pristane had reduced numbers of inflammatory mononuclear phagocytes and cNK cells in their kidneys, resembling untreated control mice. Systemically, circulating B cells and monocytes (CD115+Ly6C+) were reduced in pristane-treated Ifnlr1−/− mice. The present study supports a significant role for type III interferons in the pathogenesis of pristane-induced murine autoimmunity as well as in systemic and renal inflammation. Although the absence of type III interferon receptors does not completely prevent the development of autoantibodies, type III interferon signaling accelerates the development of autoimmunity and promotes a pro-inflammatory environment in autoimmune-prone hosts.

Neural stem cells traffic functional mitochondria via extracellular vesicles.

Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.

Differential Changes in Inflammatory Mononuclear Phagocyte and T-Cell Profiles within Psoriatic Skin during Treatment with Guselkumab vs. Secukinumab

Cellular sources of IL-23 and IL-17A driving skin inflammation in psoriasis remain unclear. Using high-dimensional unsupervised flow cytometry analysis, mononuclear phagocytes and T cells were examined in the same lesions of patients before and during guselkumab (IL-23p19 blocker) or secukinumab (IL-17A blocker) treatment. Among CD11c+HLA-DR+ mononuclear phagocytes, CD64brightCD163-CD14brightCD1c-CD1a‒ inflammatory monocyte‒like cells were the predominant IL-23-producing cells and, together with CD64-CD163-CD14-IL-23p19-TNF-α+ inflammatory dendritic cell‒like cells, were increased in lesional compared with those in nonlesional skin taken from the same patient. Within T cells, CD8+CD49a+ and/or CD103+ tissue-resident memory T cells, CD4+CD25+FoxP3+ regulatory T cells, and CD4+CD49a-CD103- T cells were increased. Moreover, CD4+CD49a-CD103- T cells and the relatively rare CD8+ memory T cells equally contributed to IL-17A production. Both treatments decreased the frequencies of inflammatory monocyte‒like, inflammatory dendritic cell‒like, and CD4+CD49a-CD103- T cells. In contrast, guselkumab reduced memory T cells while maintaining regulatory T cells and vice versa for secukinumab. Neither drug modified the frequencies of IL-17A+IL‒17F+/- CD4+ or CD8+ T cells. This study reveals the identity of the major IL-23+ mononuclear phagocyte and IL-17+ T-cell subsets in psoriatic skin lesions and paves the way for a better understanding of the mode of action of drugs targeting the IL-23/IL-17A pathway in psoriasis.

Monocytes in sarcoidosis are potent tumour necrosis factor producers and predict disease outcome.

BackgroundPulmonary sarcoidosis is an inflammatory disease characterised by granuloma formation and heterogeneous clinical outcome. Tumour necrosis factor (TNF) is a pro-inflammatory cytokine contributing to granuloma formation and high levels of TNF have been shown to associate with progressive disease. Mononuclear phagocytes (MNPs) are potent producers of TNF and highly responsive to inflammation. In sarcoidosis, alveolar macrophages have been well studied. However, MNPs also include monocytes/monocyte-derived cells and dendritic cells, which are poorly studied in sarcoidosis, despite their central role in inflammation.ObjectiveTo determine the role of pulmonary monocyte-derived cells and dendritic cells during sarcoidosis.MethodsWe performed in-depth phenotypic, functional and transcriptomic analysis of MNP subsets from blood and bronchoalveolar lavage (BAL) fluid from 108 sarcoidosis patients and 30 healthy controls. We followed the clinical development of patients and assessed how the repertoire and function of MNP subsets at diagnosis correlated with 2-year disease outcome.ResultsMonocytes/monocyte-derived cells were increased in blood and BAL of sarcoidosis patients compared to healthy controls. Interestingly, high frequencies of blood intermediate monocytes at time of diagnosis associated with chronic disease development. RNA sequencing analysis showed highly inflammatory MNPs in BAL of sarcoidosis patients. Furthermore, frequencies of BAL monocytes/monocyte-derived cells producing TNF without exogenous stimulation at time of diagnosis increased in patients that were followed longitudinally. In contrast to alveolar macrophages, the frequency of TNF-producing BAL monocytes/monocyte-derived cells at time of diagnosis was highest in sarcoidosis patients that developed progressive disease.ConclusionOur data show that pulmonary monocytes/monocyte-derived cells are highly inflammatory and can be used as a predictor of disease outcome in sarcoidosis patients.

Single-Cell Analysis of Crohn's Disease Lesions Identifies a Pathogenic Cellular Module Associated with Resistance to Anti-TNF Therapy.

Clinical benefits of cytokine blockade in ileal Crohn's disease (iCD) are limited to a subset of patients. Here, we applied single-cell technologies to iCD lesions to address whether cellular heterogeneity contributes to treatment resistance. We found that a subset of patients expressed a unique cellular module in inflamed tissues that consisted of IgG plasma cells, inflammatory mononuclear phagocytes, activated Tcells, and stromal cells, which we named the GIMATS module. Analysis of ligand-receptor interaction pairs identified a distinct network connectivity that likely drives the GIMATS module. Strikingly, the GIMATS module was also present in a subset of patients in four independent iCD cohorts (n= 441), and its presence at diagnosis correlated with failure to achieve durable corticosteroid-free remission upon anti-TNF therapy. These results emphasize the limitations of current diagnostic assays and the potential for single-cell mapping tools to identify novel biomarkers of treatment response and tailored therapeutic opportunities.

Fbxw7 increases CCL2/7 in CX3CR1hi macrophages to promote intestinal inflammation.

Resident and inflammatory mononuclear phagocytes (MPh) with functional plasticity in the intestine are critically involved in the pathology of Inflammatory Bowel Diseases (IBD), in which the mechanism remains incompletely understood. In the present study, we found that increased expression of E3 ligase FBXW7 in the inflamed intestine was significantly correlated to IBD severity in both human diseases and mice model. Myeloid-Fbxw7 deficiency protected mice from dextran sodium sulfate (DSS) and 2,6,4-trinitrobenzene sulfonic acid (TNBS) induced colitis. Fbxw7 deficiency resulted in decreased production of chemokines CCL2 and CCL7 by colonic CX3CR1hi resident macrophages and reduced accumulation of CX3CR1int pro-inflammatory MPh in colitis colon tissue. Mice received AAV-shFbxw7 administration showed significantly improved survival rate and alleviated colitis. Mechanisms screening demonstrated that FBXW7 suppresses H3K27me3 modification and promotes Ccl2 and Ccl7 expression via degradation of histone-lysine N-methyltransferase EZH2 in macrophages. Taken together, our results indicate that FBXW7 degrades EZH2 and increases Ccl2/Ccl7 in CX3CR1hi macrophages, which promotes the recruiting CX3CR1int pro-inflammatory MPh into local colon tissues with colitis. Targeting FBXW7 might represent a potential therapeutic approach for intestine inflammation intervention.

Human Lung Mononuclear Phagocytes in Health and Disease.

The lungs are vulnerable to attack by respiratory insults such as toxins, allergens, and pathogens, given their continuous exposure to the air we breathe. Our immune system has evolved to provide protection against an array of potential threats without causing collateral damage to the lung tissue. In order to swiftly detect invading pathogens, monocytes, macrophages, and dendritic cells (DCs)—together termed mononuclear phagocytes (MNPs)—line the respiratory tract with the key task of surveying the lung microenvironment in order to discriminate between harmless and harmful antigens and initiate immune responses when necessary. Each cell type excels at specific tasks: monocytes produce large amounts of cytokines, macrophages are highly phagocytic, whereas DCs excel at activating naïve T cells. Extensive studies in murine models have established a division of labor between the different populations of MNPs at steady state and during infection or inflammation. However, a translation of important findings in mice is only beginning to be explored in humans, given the challenge of working with rare cells in inaccessible human tissues. Important progress has been made in recent years on the phenotype and function of human lung MNPs. In addition to a substantial population of alveolar macrophages, three subsets of DCs have been identified in the human airways at steady state. More recently, monocyte-derived cells have also been described in healthy human lungs. Depending on the source of samples, such as lung tissue resections or bronchoalveolar lavage, the specific subsets of MNPs recovered may differ. This review provides an update on existing studies investigating human respiratory MNP populations during health and disease. Often, inflammatory MNPs are found to accumulate in the lungs of patients with pulmonary conditions. In respiratory infections or inflammatory diseases, this may contribute to disease severity, but in cancer patients this may improve clinical outcomes. By expanding on this knowledge, specific lung MNPs may be targeted or modulated in order to attain favorable responses that can improve preventive or treatment strategies against respiratory infections, lung cancer, or lung inflammatory diseases.

Atypical chemokine receptor 1 deficiency reduces atherogenesis in ApoE-knockout mice.

Atypical chemokine receptor 1 (Ackr1; previously known as the Duffy antigen receptor for chemokines or Darc) is thought to regulate acute inflammatory responses in part by scavenging inflammatory CC and CXC chemokines; however, evidence for a role in chronic inflammation has been lacking. Here we investigated the role of Ackr1 in chronic inflammation, in particular in the setting of atherogenesis, using the apolipoprotein E-deficient (ApoE(-/-)) mouse model. Ackr1(-/-)ApoE(-/-) and Ackr1(+/+)ApoE(-/-) littermates were obtained by crossing ApoE(-/-) mice and Ackr1(-/-) mice on a C57BL/6J background. Ackr1 (+/+)ApoE(-/-)mice fed a Western diet up-regulated Ackr1 expression in the aorta and had markedly increased atherosclerotic lesion size compared with Ackr1(-/-)ApoE(-/-) mice. This difference was observed in both the whole aorta and the aortic root in both early and late stages of the model. Ackr1 deficiency did not affect serum cholesterol levels or macrophage, collagen or smooth muscle cell content in atherosclerotic plaques, but significantly reduced the expression of Ccl2 and Cxcl1 in the whole aorta of ApoE(-/-) mice. In addition, Ackr1 deficiency resulted in a modest decrease in T cell subset frequency and inflammatory mononuclear phagocyte content in aorta and blood in the model. Ackr1 deficiency appears to be protective in the ApoE knockout model of atherogenesis, but it is associated with only modest changes in cytokine and chemokine expression as well as T-cell subset frequency and inflammatory macrophage content.

Macrophage Phenotype Controls Long-Term AKI Outcomes—Kidney Regeneration versus Atrophy

The mechanisms that determine full recovery versus subsequent progressive CKD after AKI are largely unknown. Because macrophages regulate inflammation as well as epithelial recovery, we investigated whether macrophage activation influences AKI outcomes. IL-1 receptor-associated kinase-M (IRAK-M) is a macrophage-specific inhibitor of Toll-like receptor (TLR) and IL-1 receptor signaling that prevents polarization toward a proinflammatory phenotype. In postischemic kidneys of wild-type mice, IRAK-M expression increased for 3 weeks after AKI and declined thereafter. However, genetic depletion of IRAK-M did not affect immunopathology and renal dysfunction during early postischemic AKI. Regarding long-term outcomes, wild-type kidneys regenerated completely within 5 weeks after AKI. In contrast, IRAK-M(-/-) kidneys progressively lost up to two-thirds of their original mass due to tubule loss, leaving atubular glomeruli and interstitial scarring. Moreover, M1 macrophages accumulated in the renal interstitial compartment, coincident with increased expression of proinflammatory cytokines and chemokines. Injection of bacterial CpG DNA induced the same effects in wild-type mice, and TNF-α blockade with etanercept partially prevented renal atrophy in IRAK-M(-/-) mice. These results suggest that IRAK-M induction during the healing phase of AKI supports the resolution of M1 macrophage- and TNF-α-dependent renal inflammation, allowing structural regeneration and functional recovery of the injured kidney. Conversely, IRAK-M loss-of-function mutations or transient exposure to bacterial DNA may drive persistent inflammatory mononuclear phagocyte infiltrates, which impair kidney regeneration and promote CKD. Overall, these results support a novel role for IRAK-M in the regulation of wound healing and tissue regeneration.

Contrasting responses of Kupffer cells and inflammatory mononuclear phagocytes to biliary obstruction in a mouse model of cholestatic liver injury

Biliary obstruction and cholestasis are serious complications of many liver diseases. Although resident hepatic macrophages (Kupffer cells) are frequently implicated in disease progression, most studies fail to differentiate the contribution of Kupffer cells and inflammatory mononuclear phagocytes (iMNPs) that infiltrate the liver subsequent to obstruction. This study was undertaken to examine the roles and potential interactions of these two disparate mononuclear phagocyte populations in hepatic injury attending cholestasis. Female, C57Bl/6 mice were injected with magnetic beads on day 3 prior to sham operation or bile duct ligation (BDL) to facilitate subsequent Kupffer cell isolation. Three days post-surgery, animals were euthanized, and bead-containing Kupffer cells and iMNPs were separated, purified and analysed. To examine the ability of Kupffer cells to modulate iMNP activity, iMNPs were isolated from the livers of intact and Kupffer cell-depleted mice on day 3 post-surgery and compared. Purified Kupffer cells and iMNP populations obtained from BDL mice exhibited heterogeneous morphologies rendering them visually indistinguishable. iMNPs, however, were characterized by the increased expression of Ly-6C and CD11b and the elevated production of chemokines/cytokines characteristic of inflammatory cells. In the absence of Kupffer cells, iMNPs immigrating to the liver following BDL exhibited significant decreases in CD11b and Ly-6C expression, and in pro-inflammatory chemokine/cytokine production. Kupffer cells and iMNPs exhibit disparate biological responses to biliary obstruction and cholestasis. Kupffer cells play a key role in regulating iMNP influx and activity.

Kupffer cells regulate the biological response of inflammatory mononuclear phagocytes to biliary obstruction in mice. (117.6)

Abstract Background: Biliary obstruction is a serious complication of liver diseases. Most studies fail to differentiate the contribution of resident hepatic macrophages (Kupffer cells, KC) and inflammatory mononuclear phagocytes (iMNP) that infiltrate the liver subsequent to injury. This study was undertaken to examine the distinct roles and interactions of these mononuclear phagocyte populations in liver injury attending cholestasis. Methods: Female, C57Bl/6 mice were injected with magnetic beads 3 days prior to sham operation or bile duct ligation (BDL) to facilitate subsequent KC isolation. Three days post-surgery, bead-containing KC and iMNP were separated, purified and analyzed. To examine the ability of KC to modulate iMNP activity, iMNP were isolated from intact and KC-depleted livers on day 3 post-surgery and compared. Results: Following BDL, iMNP exhibited increased expression of Ly-6C and elevated production of chemokines/ cytokines compared to KC. In the absence of KC, iMNP exhibited significant decreases in Ly-6C expression and inflammatory chemokine/cytokine production. Conclusions: KC play a key role in regulating the influx and biological response of iMNP to biliary obstruction and cholestasis.

Immunodetection of cyclooxygenase-2 (COX-2) is restricted to tissue macrophages in normal rat liver and to recruited mononuclear phagocytes in liver injury and cholangiocarcinoma

It has been suggested that cyclooxygenase-2 (COX-2)-mediated prostaglandin synthesis is associated with liver inflammation and carcinogenesis. The aim of this study is to identify the cellular source of COX-2 expression in different stages, from acute liver injury through liver fibrosis to cholangiocarcinoma (CC). We induced in rats acute and “chronic” liver injury (thioacetamide (TAA) or carbon tetrachloride (CCl4)) and CC development (TAA) and assessed COX-2 gene expression in normal and damaged liver tissue by RT-PCR of total RNA. The cellular localization of COX-2 protein in liver tissue was analyzed by immunohistochemistry as well as in isolated rat liver cells by Western blotting. The findings were compared with those obtained in human cirrhotic liver tissue. The specificity of the antibodies was tested by 2-DE Western blot and mass spectrometric identification of the positive protein spots. RT-PCR analysis of total RNA revealed an increase of hepatic COX-2 gene expression in acutely as well as “chronically” damaged liver. COX-2-protein was detected in those ED1+/ED2+ cells located in the non-damaged tissue (resident tissue macrophages). In addition COX-2 positivity in inflammatory mononuclear phagocytes (ED1+/ED2−), which were also present within the tumoral tissue was detected. COX-2 protein was clearly detectable in isolated Kupffer cells as well as (at lower level) in isolated “inflammatory” macrophages. Similar results were obtained in human cirrhotic liver. COX-2 protein is constitutively detectable in liver tissue macrophages. Inflammatory mononuclear phagocytes contribute to the increase of COX-2 gene expression in acute and chronic liver damage induced by different toxins and in the CC microenvironment.Electronic supplementary materialThe online version of this article (doi:10.1007/s00418-011-0889-9) contains supplementary material, which is available to authorized users.

Different physiology of interferon-α/-γ in models of liver regeneration in the rat

Liver regeneration may take place after liver injury through replication of hepatocytes or hepatic progenitor cells called oval cells. Interferons (IFN) are natural cytokines with pleiotrophic effects including antiviral and antiproliferative actions. No data are yet available on the physiology and cellular source of natural IFNs during liver regeneration. To address this issue, we have analyzed the levels and biologic activities of IFN-α/IFN-γ in two models of partial hepatectomy. After 2/3rd partial hepatectomy (PH), hepatic levels of IFN-α and IFN-γ declined transiently in contrast to a transient increase of the IFN-γ serum level. After administration of 2-acetylaminofluorene and partial hepatectomy (AAF/PH model), however, both IFN-α and IFN-γ expression were up-regulated in regenerating livers. Again, the IFN-γ serum level was transiently increased. Whereas hepatic IFN-γ was up-regulated early (day 1–5), but not significantly, in the AAF/PH model, IFN-α was significantly up-regulated at later time points in parallel to the peak of oval cell proliferation (days 7–9). Biological activity of IFN-α was shown by activation of IFN-α-specific signal transduction and induction of IFN-α specific-gene expression. We found a significant infiltration of the liver with inflammatory monocyte-like mononuclear phagocytes (MNP) concomitant to the frequency of oval cells. We localized IFN-α production only in MNPs, but not in oval cells. These events were not observed in normal liver regeneration after standard PH. We conclude that IFN-γ functions as an acute-phase cytokine in both models of liver regeneration and may constitute a systemic component of liver regeneration. IFN-α was increased only in the AAF/PH model, and was associated with proliferation of oval cells. However, oval cells seem not to be the source of IFN-α. Instead, inflammatory MNP infiltrating AAF/PH-treated livers produce IFN-α. These inflammatory MNPs may be involved in the regulation of the oval cell compartment through local expression of cytokines, including IFN-α.

Xanthine Oxidoreductase Promotes the Inflammatory State of Mononuclear Phagocytes through Effects on Chemokine Expression, Peroxisome Proliferator-activated Receptor-γ Sumoylation, and HIF-1α

The protective effects of pharmacological inhibitors of xanthine oxidoreductase (XOR) have implicated XOR in many inflammatory diseases. Nonetheless, the role played by XOR during inflammation is poorly understood. We previously observed that inhibition of XOR within the inflammatory mononuclear phagocytes (MNP) prevented neutrophil recruitment during adoptive transfer demonstrating the role of XOR in MNP-mediated neutrophil recruitment. To further explore the role of XOR in the inflammatory state of MNP, we studied MNP isolated from inflammatory lungs combined with analyses of MNP cell lines. We demonstrated that XOR activity was increased in inflammatory MNP following insufflation of Th-1 cytokines in vivo and that activity was specifically increased by MNP differentiation. Inhibition of XOR reduced levels of CINC-1 secreted by MNP. Expression of peroxisome proliferator-activated receptor γ (PPARγ) in purified rat lung MNP and MNP cell lines reflected both the presence of PPARγ isoforms and PPARγ SUMOylation, and XOR inhibitors increased levels of SUMO-PPARγ in MNP cell lines. Both ectopic overexpression of XOR cDNA and uric acid supplementation reduced SUMO-PPARγ in MNP cells. Levels of the M2 markers CD36, CD206, and arginase-1 were modulated by uric acid and oxonic acid, whereas siRNA to SUMO-1 or PIAS-1 also reduced arginase-1 in RAW264.7 cells. We also observed that HIF-1α was increased by XOR inhibitors in inflammatory MNP and in MNP cell lines. These data demonstrate that XOR promotes the inflammatory state of MNP through effects on chemokine expression, PPARγ SUMOylation, and HIF-1α and suggest that strategies for inhibiting XOR may be valuable in modulating lung inflammatory disorders.

Dynamics of immune granuloma formation in a Leishmania braziliensis‐induced self‐limiting cutaneous infection in the primate Macaca mulatta

In order to unravel the physiopathology of leishmaniasis in humans, it is necessary to better understand how Leishmania are able to survive for years within immunologically active granulomas. In the present study, we used a macaque (Macaca mulatta) model of infection with Leishmania braziliensis as a means of assessing the usefulness of this primate system. This model more closely mirrors human protective immunity to Leishmania than the murine model; therefore, we used it to study the host inflammatory granulomatous response involved in the control of cutaneous leishmaniasis. Infected primates developed localized long-term skin ulcerations, but complete spontaneous clinical healing occurred in all infected animals. The infection induced the recruitment and activation of inflammatory mast cells, granulocytes, mononuclear phagocytes, and lymphocytes at the site of infection. During the acute reaction, polymorphonuclear leukocytes were more prominent than other cell types and apparently destroyed many parasites; macrophages then rapidly engulfed dying neutrophils together with their parasitic cargo. In the chronic phase, persisting parasites induced a typical T helper (Th) cytokine, type 1-mediated, immunity-induced granulomatous reaction. By this time, more or less differentiated macrophage accumulations were found, and these evolved to become mature tissue granulomas consisting of all the specific cell types found within human granulomas. In the healing stage, fibroblasts proliferated at the periphery and finally invaded the granulomas with fibrotic substitution. These findings point to the feasibility of using this model to elucidate the potentially disabling Th1-cell mechanisms that may eventually render the host granulomatous response inadequate for fighting L. braziliensis infections.

Lung-specific overexpression of CC chemokine ligand (CCL2) 2 enhances the host defense to Streptococcus pneumoniae infection in mice: role of the CCL2-CCR2 axis

Background: Mononuclear phagocytes are critical components of the lung innate host defense to inhaled bacterial pathogens. The monocyte chemotactic protein CCL2 plays a pivotal role in inflammatory mononuclear phagocyte recruitment. Here, we tested the hypothesis that increased CCL2-dependent mononuclear phagocyte recruitment would improve the lung innate host defense to infection with Streptococcus pneumoniae.

Lung-Specific Overexpression of CC Chemokine Ligand (CCL) 2 Enhances the Host Defense to Streptococcus pneumoniae Infection in Mice: Role of the CCL2-CCR2 Axis

Mononuclear phagocytes are critical components of the innate host defense of the lung to inhaled bacterial pathogens. The monocyte chemotactic protein CCL2 plays a pivotal role in inflammatory mononuclear phagocyte recruitment. In this study, we tested the hypothesis that increased CCL2-dependent mononuclear phagocyte recruitment would improve lung innate host defense to infection with Streptococcus pneumoniae. CCL2 transgenic mice that overexpress human CCL2 protein in type II alveolar epithelial cells and secrete it into the alveolar air space showed a similar proinflammatory mediator response and neutrophilic alveolitis to challenge with S. pneumoniae as wild-type mice. However, CCL2 overexpressing mice showed an improved pneumococcal clearance and survival compared with wild-type mice that was associated with substantially increased lung mononuclear phagocyte subset accumulations upon pneumococcal challenge. Surprisingly, CCL2 overexpressing mice developed bronchiolitis obliterans upon pneumococcal challenge. Application of anti-CCR2 Ab MC21 to block the CCL2-CCR2 axis in CCL2 overexpressing mice, though completely abrogating bronchiolitis obliterans, led to progressive pneumococcal pneumonia. Collectively, these findings demonstrate the importance of the CCL2-CCR2 axis in the regulation of both the resolution/repair and remodelling processes after bacterial challenge and suggest that overwhelming innate immune responses may trigger bronchiolitis obliterans formation in bacterial lung infections.

Repair after cholestatic liver injury correlates with neutrophil infiltration and matrix metalloproteinase 8 activity

Although timely surgical treatment of liver disease can interrupt inflammation and reduce fibrosis, the mechanisms of repair are unknown. We questioned whether these mechanisms of repair include changes in the inflammatory infiltrate and associated biological activity of matrix metalloproteinases (MMPs) 8 and 2. Rats (n >or= 3) underwent biliary ductal suspension for 7 days followed by decompression. Livers were collected after 7 days of obstruction (d0) and after 2, 5, and 7 days of repair (d2, d5, d7, respectively), and assessed morphometrically for collagen, polymorphonuclear cells (PMNs), Kupffer cells (KCs), and inflammatory mononuclear phagocytes (MNPs). In situ zymography was performed by using fluorogenic substrates for MMP-8 and MMP-2 to spatially localize enzymatic activity. Cholestatic injury resulted in significantly elevated (P <or= .001) collagen deposition (3-fold), and elevated numbers of MNPs (10-fold), KCs (5-fold), and PMNs (4-fold), compared with shams. PMNs remained elevated through d7, while collagen deposition, KCs, and MNPs returned to sham levels by d2. In situ zymography showed no significant changes in MMP-2 activity after cholestatic injury and repair. MMP-8 activity was significantly (P <or= .05) elevated only during repair. Activity was localized to fibrotic portal triads containing PMNs. Cholestatic injury results in increased fibrosis, MNPs, KCs, and PMNs but no MMP-2 or MMP-8 activity. Biliary decompression results in increased MMP-8 activity co-localized to areas of portal fibrosis and PMN accumulation. We conclude that secretion of MMP-8 by neutrophils may play a critical role in resolving the fibrotic scar generated during cholestasis.

The Inflammatory versus Constitutive Trafficking of Mononuclear Phagocytes into the Alveolar Space of Mice Is Associated with Drastic Changes in Their Gene Expression Profiles

Mononuclear phagocytes enter the lungs both constitutively to maintain alveolar macrophage and dendritic cell homeostasis, as well as during lung inflammation, where the role of these cells is less well defined. We used a transgenic mouse strain (CX3CR1(+/GFP)) that harbors a GFP label in circulating monocytes to identify and sort these cells from the vascular and alveolar compartments under both constitutive and acute lung inflammatory conditions. Using nylon arrays combined with real-time RT-PCR for gene expression profiling, we found that flow-sorted, highly purified mononuclear phagocytes recruited to acutely inflamed mouse lungs showed strongly up-regulated mRNA levels of the neutrophil chemoattractants KC, MIP-2, and IP-10, which contrasted with alveolar mononuclear phagocytes that immigrated in steady state. Similar observations were made for the lysosomal cathepsins B, L, and K being strongly up-regulated in mononuclear phagocytes upon recruitment to inflamed lungs but not during constitutive alveolar immigration. Inflammatory elicited mononuclear phagocytes also demonstrated significantly increased mRNA levels of the cytokine TNF-alpha and the PRR-associated molecules CD14, TLR4, and syndecan-4. Together, inflammatory elicited mononuclear phagocytes exhibit strongly increased neutrophil chemoattractants, lysosomal proteases, and LPS signaling mRNA transcripts, suggesting that these cells may play a major role in acute lung inflammatory processes.

Receptor for Advanced Glycation End Products and Its Ligands: A Journey from the Complications of Diabetes to Its Pathogenesis

Many studies have suggested that the expression of RAGE (receptor for advanced glycation end products) is upregulated in human tissues susceptible to the long-term complications of diabetes. From the kidneys to the macrovessels of the aorta, RAGE expression is upregulated in a diverse array of cell types, from glomerular epithelial cells (podocytes) to endothelial cells, vascular smooth muscle cells, and inflammatory mononuclear phagocytes and lymphocytes. Although RAGE was first described as a receptor for advanced glycation end products (AGEs), the key finding that RAGE was also a signaling receptor for proinflammatory S100/calgranulins and amphoterin, led to the premise that even in euglycemia, ligand-RAGE interaction propagated inflammatory mechanisms linked to chronic cellular perturbation and tissue injury. Indeed, such considerations suggested that RAGE might even participate in the pathogenesis of type 1 diabetes. Our studies have shown that pharmacological and/or genetic deletion/mutation of the receptor attenuates the development of hyperglycemia in NOD mice; in mice with myriad complications of diabetes, interruption of ligand-RAGE interaction prevents or delays the chronic complications of the disease in both macro- and microvessel structures. Taken together, these findings suggest that RAGE is "at the right place and time" to contribute to the pathogenesis of diabetes and it complications. Studies are in progress to test the premise that antagonism of this interaction is a logical strategy for the prevention and treatment of diabetes.