Induction of Alternatively Activated Macrophages Enhances Pathogenesis during Severe Acute Respiratory Syndrome Coronavirus Infection

Infection with severe acute respiratory syndrome coronavirus (SARS-CoV) causes acute lung injury (ALI) that often leads to severe lung disease. A mouse model of acute SARS-CoV infection has been helpful in understanding the host response to infection; however, there are still unanswered questions concerning SARS-CoV pathogenesis. We have shown that STAT1 plays an important role in the severity of SARS-CoV pathogenesis and that it is independent of the role of STAT1 in interferon signaling. Mice lacking STAT1 have greater weight loss, severe lung pathology with pre-pulmonary-fibrosis-like lesions, and an altered immune response following infection with SARS-CoV. We hypothesized that STAT1 plays a role in the polarization of the immune response, specifically in macrophages, resulting in a worsened outcome. To test this, we created bone marrow chimeras and cell-type-specific knockouts of STAT1 to identify which cell type(s) is critical to protection from severe lung disease after SARS-CoV infection. Bone marrow chimera experiments demonstrated that hematopoietic cells are responsible for the pathogenesis in STAT1^−/− mice, and because of an induction of alternatively activated (AA) macrophages after infection, we hypothesized that the AA macrophages were critical for disease severity. Mice with STAT1 in either monocytes and macrophages (LysM/STAT1) or ciliated lung epithelial cells (FoxJ1/STAT1) deleted were created. Following infection, LysM/STAT1 mice display severe lung pathology, while FoxJ1/STAT1 mice display normal lung pathology. We hypothesized that AA macrophages were responsible for this STAT1-dependent pathology and therefore created STAT1/STAT6^−/− double-knockout mice. STAT6 is essential for the development of AA macrophages. Infection of the double-knockout mice displayed a lack of lung disease and prefibrotic lesions, suggesting that AA macrophage production may be the cause of STAT1-dependent lung disease. We propose that the control of AA macrophages by STAT1 is critical to regulating immune pathologies and for protection from long-term progression to fibrotic lung disease in a mouse model of SARS-CoV infection.     

Infiltration of Alternatively Activated Macrophages in Cancer Tissue Is Associated with MDSC and Th2 Polarization in Patients with Esophageal Cancer

Myeloid derived suppressor cells (MDSCs) expand in cancer bearing hosts and contribute to tumor immune evasion. M2 macrophages constitute a major cellular component of cancer-related inflammation. However, the correlation between circulating MDSCs and infiltrating M2 macrophages in tumor tissues from patients with esophageal cancer (ECA), and its potential relationship with the polarization of Th2 cells remain unclear. In the present study, we showed the level of MDSCs in PBMC and Arg1 in plasma were significantly elevated in ECA patients, and the increased ratio of MDSC in PBMC was closely related to the expression of CD163 in cancer tissues. In addition, the ECA patients exhibited remarkable increases in the mRNA levels of IL-4 and GATA3, as well as the protein levels of IL-13 and IL-6, but IFN-γ and IL-12 in peripheral blood were decreased. Our data indicate that the increased Th2 cytokines are associated with MDSCs and M2 macrophages polarization, and foster the infiltration of CD163⁺M2 macrophages in cancer tissues, which promote the formation of immunosuppressive microenvironment in ECA patients.     

Ly6Chigh Monocytes Become Alternatively Activated Macrophages in Schistosome Granulomas with Help from CD4+ Cells

Alternatively activated macrophages (AAM) that accumulate during chronic T helper 2 inflammatory conditions may arise through proliferation of resident macrophages or recruitment of monocyte-derived cells. Liver granulomas that form around eggs of the helminth parasite Schistosoma mansoni require AAM to limit tissue damage. Here, we characterized monocyte and macrophage dynamics in the livers of infected CX3CR1^GFP/+ mice. CX₃CR1-GFP⁺ monocytes and macrophages accumulated around eggs and in granulomas during infection and upregulated PD-L2 expression, indicating differentiation into AAM. Intravital imaging of CX₃CR1-GFP⁺ Ly6C^low monocytes revealed alterations in patrolling behavior including arrest around eggs that were not encased in granulomas. Differential labeling of CX₃CR1-GFP⁺ cells in the blood and the tissue showed CD4⁺ T cell dependent accumulation of PD-L2⁺ CX₃CR1-GFP⁺ AAM in the tissues as granulomas form. By adoptive transfer of Ly6C^high and Ly6C^low monocytes into infected mice, we found that AAM originate primarily from transferred Ly6C^high monocytes, but that these cells may transition through a Ly6C^low state and adopt patrolling behavior in the vasculature. Thus, during chronic helminth infection AAM can arise from recruited Ly6C^high monocytes via help from CD4⁺ T cells.     

白介素-6细胞因子家族新成员：心肌营养素-1

心肌营养素-1(cardiotrophin-1, CT-1)是一个新发现的白介素-6家族细胞因子,小鼠CT-1的mRNA长约1.4 kb,编码蛋白由203个氨基酸组成,以gp130和LIFR为受体.CT-1对心肌细胞既有肥大诱导作用,又有保护作用;能改变交感神经元的递质表型,促进多巴胺神经元、睫状神经元和运动神经元的存活;还能抑制骨髓白血病细胞M1的生长;诱导肝细胞急性相反应;小鼠腹腔注射给药可增加血小板、红细胞记数和血红蛋白浓度.     

Interleukin-6, a Major Cytokine in the Central Nervous System

Interleukin-6 (IL-6) is a cytokine originally identified almost 30 years ago as a B-cell differentiation factor, capable of inducing the maturation of B cells into antibody-producing cells. As with many other cytokines, it was soon realized that IL-6 was not a factor only involved in the immune response, but with many critical roles in major physiological systems including the nervous system. IL-6 is now known to participate in neurogenesis (influencing both neurons and glial cells), and in the response of mature neurons and glial cells in normal conditions and following a wide arrange of injury models. In many respects, IL-6 behaves in a neurotrophin-like fashion, and seemingly makes understandable why the cytokine family that it belongs to is known as neuropoietins. Its expression is affected in several of the main brain diseases, and animal models strongly suggest that IL-6 could have a role in the observed neuropathology and that therefore it is a clear target of strategic therapies.     

The Predominance of Alternatively Activated Macrophages Following Challenge with Cell Wall Peptide-Polysaccharide After Prior Infection with Sporothrix schenckii

Sporotrichosis is a subcutaneous mycosis that is caused by the dimorphic fungus Sporothrix schenckii. This disease generally occurs within the skin and subcutaneous tissues, causing lesions that can spread through adjacent lymphatic vessels and sometimes leading to systemic diseases in immunocompromised patients. Macrophages are crucial for proper immune responses against a variety of pathogens. Furthermore, macrophages can play different roles in response to different microorganisms and forms of activation, and they can be divided into “classic” or “alternatively” activated populations, as also known as M1 and M2 macrophages. M1 cells can lead to tissue injury and contribute to pathogenesis, whereas M2 cells promote angiogenesis, tissue remodeling, and repair. The aim of this study was to investigate the roles of M1 and M2 macrophages in a sporotrichosis model. Toward this end, we performed phenotyping of peritoneal exudate cells and evaluated the concomitant production of several immunomediators, including IL-12, IL-10, TGF-β, nitric oxide, and arginase-I activity, which were stimulated ex vivo with cell wall peptide-polysaccharide. Our results showed the predominance of the M2 macrophage population, indicated by peaks of arginase-I activity as well as IL-10 and TGF-β production during the 6th and 8th weeks after infection. These results were consistent with cellular phenotyping that revealed increases in CD206-positive cells over this period. This is the first report of the participation of M2 macrophages in sporotrichosis infections.     

Interleukin-1 Hyperproduction by In Vitro Activated Peripheral Macrophages from Cerebellar Mutant Mice

Several mutations in mice produce complex patterns of neuronal degeneration of the cerebellum and of its afferent pathways. In the staggerer (sg/sg) mutant, atrophy of the lymphoid organs and immunological abnormalities have been described. To search for a possible link between the neurological and the immune disorders in this mutant, we studied the production by its peripheral macrophages of interleukin-1 (IL-1), which roles in both immune and nervous systems are well established. Suspensions of peritoneal and/or spleen macrophages from mutants and their appropriate controls were stimulated in vitro by lipopolysaccharide. Northern and dot blots, performed with murine IL-1 cDNA probes, revealed a clear-cut hyperexpression of IL-1 mRNA in staggerer macrophages. An IL-1 bioassay using the IL-1-responsive D10.G4 cell line also revealed a sixfold increase of IL-1 activity in the macrophage supernatants of staggerer mutant mice. The hyperproduction was found in 3-week to 1-year-old staggerer and also in heterozygous (+/sg) mice. A similar phenomenon existed in cerebellar mutants lurcher, Purkinje cell degeneration (pcd), and to a lesser extent reeler and wobbler, but was absent in the neurological mutants weaver, jimpy, and motor end plate disease (medH). These observations establish that in several point mutations in mice, central nervous degeneration is associated with dysregulation of IL-1 production by peripheral macrophages.     

巨噬细胞的极化及其意义

表型可变性和功能多样性是单个核吞噬细胞的重要特征。近年来巨噬细胞的极化受到关注。一般认为极化巨噬细胞是单核细胞活化后一系列功能状态两个极端。而它的分化受到各种微环境信号的诱导与调节。极化的巨噬细胞能够进一步影响局部免疫反应,与各种因子协同作用调节病原体微生物感染结局和肿瘤免疫,参与免疫调节,组织修复重塑过程。对巨噬细胞亚型诱导因素及功能的研究将有助于了解免疫反应的调节机制。     

Upregulation of Retinal Dehydrogenase 2 in Alternatively Activated Macrophages during Retinoid-dependent Type-2 Immunity to Helminth Infection in Mice

Although the vitamin A metabolite retinoic acid (RA) plays a critical role in immune function, RA synthesis during infection is poorly understood. Here, we show that retinal dehydrogenases (Raldh), required for the synthesis of RA, are induced during a retinoid-dependent type-2 immune response elicited by Schistosoma mansoni infection, but not during a retinoid-independent anti-viral immune response. Vitamin A deficient mice have a selective defect in T_H2 responses to S. mansoni, but retained normal LCMV specific T_H1 responses. A combination of in situ imaging, intra-vital imaging, and sort purification revealed that alternatively activated macrophages (AAMφ) express high levels of Raldh2 during S. mansoni infection. IL-4 induces Raldh2 expression in bone marrow-derived macrophages in vitro and peritoneal macrophages in vivo. Finally, in vivo derived AAMφ have an enhanced capacity to induce Foxp3 expression in CD4+ cells through an RA dependent mechanism, especially in combination with TGF-β. The regulation of Raldh enzymes during infection is pathogen specific and reflects differential requirements for RA during effector responses. Specifically, AAMφ are an inducible source of RA synthesis during helminth infections and T_H2 responses that may be important in regulating immune responses.     

P2Y6 Receptor Potentiates Pro-Inflammatory Responses in Macrophages and Exhibits Differential Roles in Atherosclerotic Lesion Development

Background

P2Y₆, a purinergic receptor for UDP, is enriched in atherosclerotic lesions and is implicated in pro-inflammatory responses of key vascular cell types and macrophages. Evidence for its involvement in atherogenesis, however, has been lacking. Here we use cell-based studies and three murine models of atherogenesis to evaluate the impact of P2Y₆ deficiency on atherosclerosis.

Methodology/Principal Findings

Cell-based studies in 1321N1 astrocytoma cells, which lack functional P2Y₆ receptors, showed that exogenous expression of P2Y₆ induces a robust, receptor- and agonist-dependent secretion of inflammatory mediators IL-8, IL-6, MCP-1 and GRO1. P2Y₆-mediated inflammatory responses were also observed, albeit to a lesser extent, in macrophages endogenously expressing P2Y₆ and in acute peritonitis models of inflammation. To evaluate the role of P2Y₆ in atherosclerotic lesion development, we used P2Y₆-deficient mice in three mouse models of atherosclerosis. A 43% reduction in aortic arch plaque was observed in high fat-fed LDLR knockout mice lacking P2Y₆ receptors in bone marrow-derived cells. In contrast, no effect on lesion development was observed in fat-fed whole body P2Y₆xLDLR double knockout mice. Interestingly, in a model of enhanced vascular inflammation using angiotensin II, P2Y₆ deficiency enhanced formation of aneurysms and exhibited a trend towards increased atherosclerosis in the aorta of LDLR knockout mice.

Conclusions

P2Y₆ receptor augments pro-inflammatory responses in macrophages and exhibits a pro-atherogenic role in hematopoietic cells. However, the overall impact of whole body P2Y₆ deficiency on atherosclerosis appears to be modest and could reflect additional roles of P2Y₆ in vascular disease pathophysiologies, such as aneurysm formation.     

Phagocytosis of Cells Dying through Autophagy Evokes a Pro-Inflammatory Response in Macrophages

Autophagy as a natural part of cellular homeostasis usually takes place unnoticed by neighboring cells. However, its co-occurrence with cell death may contribute to the clearance of these dying cells by recruited phagocytes. Autophagy associated with programmed cell death has recently been reported to be essential for presentation of phoshatidylserine (PS) on the cell surface (Qu et al. 2007) that has a key role in the clearance of apoptotic cells. Recently, we have demonstrated that upon triggering cell death by autophagy in MCF-7 cells, the corpses were efficiently phagocytosed by both human macrophages and non-dying MCF-7 cells. Death as well as engulfment could be prevented by inhibiting autophagy. Based on our data, two molecular mechanisms have been proposed for the uptake of cells which die through autophagy: a PS-dependent pathway which was exclusively used by the living MCF-7 cells acting as non-professional phagocytes, and a PS-independent uptake mechanism that was active in macrophages acting as professional phagocytes. Several lines of evidence suggest that macrophages utilize calreticulin-mediated recognition, tethering, tickling and engulfment processes. Phagocytic uptake of cells dying through autophagy by macrophages leads to a pro-inflammatory response characterized by the induction and secretion of IL-6, TNFα, IL-8 and IL-10.

Addendum to:Clearance of Dying Autophagic Cells of Different Origin by Professional and Non-Professional Phagocytes

G. Petrovski, G. Zahuczky, K. Katona, G. Vereb, W. Martinet, Z. Nemes, W. Bursch and L. Fésüs

Cell Death Differ 2007;14:1117-28     

Allograft Inflammatory Factor 1 Functions as a Pro-Inflammatory Cytokine in the Oyster,Crassostrea ariakensis

The oyster Crassostrea ariakensis is an economically important bivalve species in China, unfortunately it has suffered severe mortalities in recent years caused by rickettsia-like organism (RLO) infection. Prevention and control of this disease is a priority for the development of oyster aquaculture. Allograft inflammatory factor-1 (AIF-1) was identified as a modulator of the immune response during macrophage activation and a key gene in host immune defense reaction and inflammatory response. Therefore we investigated the functions of C. ariakensis AIF-1 (Ca-AIF1) and its antibody (anti-CaAIF1) in oyster RLO/LPS-induced disease and inflammation. Ca-AIF1 encodes a 149 amino acid protein containing two typical Ca²⁺ binding EF-hand motifs and shares a 48–95% amino acid sequence identity with other animal AIF-1s. Tissue-specific expression analysis indicates that Ca-AIF1 is highly expressed in hemocytes. Significant and continuous up-regulation of Ca-AIF1 is detected when hemocytes are stimulated with RLO/LPS (RLO or LPS). Treatment with recombinant Ca-AIF1 protein significantly up-regulates the expression levels of LITAF, MyD88 and TGFβ. When anti-CaAIF1 antibody is added to RLO/LPS-challenged hemocyte monolayers, a significant reduction of RLO/LPS-induced LITAF is observed at 1.5–12 h after treatment, suggesting that interference with Ca-AIF1 can suppress the inflammatory response. Furthermore, flow cytometric analysis indicated that anti-CaAIF1 administration reduces RLO/LPS-induced apoptosis and necrosis rates of hemocytes. Collectively these findings suggest that Ca-AIF1 functions as a pro-inflammatory cytokine in the oyster immune response and is a potential target for controlling RLO infection and LPS-induced inflammation.     

Ammonia Attenuates LPS-Induced Upregulation of Pro-Inflammatory Cytokine mRNA in Co-Cultured Astrocytes and Microglia

Hepatic encephalopathy (HE) is associated with cerebral microglia activation. Ammonia, a major toxin of HE, activates microglia in vitro but does not trigger pro-inflammatory cytokine synthesis. In the present study we analysed effects of ammonia on lipopolysaccharide (LPS)-induced upregulation of microglia activation and cytokine mRNA as well as on cytokine secretion in mono-cultured microglia and co-cultured astrocytes and microglia. In mono-cultured microglia LPS (100 ng/ml, 18 h) strongly elevated mRNA levels of the microglia activation marker CD14 and the pro-inflammatory cytokines IL-1α/β, IL-6 and TNF-α. NH₄Cl (5 mmol/l) had no effect on LPS-induced upregulation of CD14, IL-1α/β and IL-6 mRNA but enhanced LPS-induced upregulation of TNF-α mRNA in mono-cultured microglia. In co-cultured astrocytes and microglia, however, LPS-induced upregulation of IL-1α/β, TNF-α, IL-6, CD14 but not of IL-10, IL-12A/B or TGFβ_1?3 mRNA was attenuated by NH₄Cl. LPS-induced upregulation of IL-1α/β, IL-6 and TNF-α was also diminished by the TGR5-ligands allopregnanolone and taurolithocholic acid in mono-cultured microglia. NH₄Cl also attenuated LPS-induced release of MCP-1, IL-6 and IL-10 in mono-cultured microglia. mRNA level of surrogate marker for microglia activation (CD14) and for the anti-inflammatory M2-type microglia (CD163, CXCL1, CXCL2) were also elevated in post mortem brain tissue taken from the fusiforme gyrus of patients with liver cirrhosis and HE. The findings suggest that ammonia attenuates LPS-induced microglia reactivity in an astrocyte-dependent way. One may speculate that these anti-inflammatory effects of ammonia may be triggered by neurosteroids derived from astrocytes and may account for absence of microglia reactivity in cerebral cortex of cirrhotic patients with HE.     

AICAR Enhances the Phagocytic Ability of Macrophages towards Apoptotic Cells through P38 Mitogen Activated Protein Kinase Activation Independent of AMP-Activated Protein Kinase

Recent studies have suggested that 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) increases macrophage phagocytosis through adenosine monophosphate-activated protein kinase (AMPK). However, little information is available on the effects of AICAR on the clearance of apoptotic cells by macrophages, known as efferocytosis, which is essential in maintaining tissue homeostasis and resolving inflammation. AICAR increased p38 MAPK activation and the phagocytosis of apoptotic cells by macrophages, which were inhibited by the p38 MAPK inhibitor, SB203580, the TGF-beta-activated kinase 1 (TAK1) inhibitor, (5Z)-7-oxozeaenol, and siRNA-mediated knock-down of p38α. AICAR increased phosphorylation of Akt, but the inhibition of PI3K/Akt activity using {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 did not affect the AICAR-induced changes in efferocytosis in macrophages. {"type":"entrez-protein","attrs":{"text":"CGS15943","term_id":"875345334","term_text":"CGS15943"}}CGS15943, a non-selective adenosine receptor antagonist, did not affect AICAR-induced changes in efferocytosis, but dipyridamole, an adenosine transporter inhibitor, diminished the AICAR-mediated increases in efferocytosis. AICAR-induced p38 MAPK phosphorylation was not inhibited by the AMPK inhibitor, compound C, or siRNA-mediated knock-down of AMPKα1. Inhibition of AMPK using compound C or 5’-iodotubercidin did not completely block AICAR-mediated increases in efferocytosis. Furthermore, AICAR also increased the removal of apoptotic neutrophils or thymocytes in mouse lungs. These results reveal a novel mechanism by which AICAR increases macrophage-mediated phagocytosis of apoptotic cells and suggest that AICAR may be used to treat efferocytosis-related inflammatory conditions.     

Unusual Water-mediated Antigenic Recognition of the Proinflammatory Cytokine Interleukin-18

The unique cytokine interleukin-18 (IL-18) acts synergistically with IL-12 to regulate T-helper 1 and 2 lymphocytes and, as such, seems to underlie the pathogenesis of various autoimmune and allergic diseases. Several anti-IL-18 agents are in clinical development, including the recombinant human antibody ABT-325, which is entering trials for autoimmune diseases. Given competing cytokine/receptor and cytokine/receptor decoy interactions, understanding the structural basis for recognition is critical for effective development of anti-cytokine therapies. Here we report three crystal structures: the murine antibody 125-2H Fab fragment bound to human IL-18, at 1.5 Å resolution; the 125-2H Fab (2.3 Å); and the ABT-325 Fab (1.5 Å). These structures, along with human/mouse IL-18 chimera binding data, allow us to make three key observations relevant to the biology and antigenic recognition of IL-18 and related cytokines. First, several IL-18 residues shift dramatically (>10 Å) upon binding 125-2H, compared with unbound IL-18 (Kato, Z., Jee, J., Shikano, H., Mishima, M., Ohki, I., Ohnishi, H., Li, A., Hashimoto, K., Matsukuma, E., Omoya, K., Yamamoto, Y., Yoneda, T., Hara, T., Kondo, N., and Shirakawa, M. (2003) Nat. Struct. Biol. 10, 966–971). IL-18 thus exhibits plasticity that may be common to its interactions with other receptors. Related cytokines may exhibit similar plasticity. Second, ABT-325 and 125-2H differ significantly in combining site character and architecture, thus explaining their ability to bind IL-18 simultaneously at distinct epitopes. These data allow us to define the likely ABT-325 epitope and thereby explain the distinct neutralizing mechanisms of both antibodies. Third, given the high 125-2H potency, 10 well ordered water molecules are trapped upon complex formation in a cavity between two IL-18 loops and all six 125-2H complementarity-determining regions. Thus, counterintuitively, tight and specific antibody binding may in some cases be water-mediated.Interleukin (IL)³ -18 is a proinflammatory cytokine that participates in the regulation of innate and acquired immunity (2, 3). IL-18 acts alone or in concert with IL-12 to amplify the induction of proinflammatory and cytotoxic mediators, such as interferon-γ. For example, in IL-18 knock-out mice, levels of interferon-γ and cytotoxic T cells decrease despite the presence of IL-12. Inhibition of IL-18 activity has been found to be beneficial in several autoimmune disease animal models (e.g. collagen-induced arthritis (4) and colitis (5)). Furthermore, IL-18 expression is dramatically increased by the chronic inflammatory state extant in human autoimmune diseases, such as rheumatoid arthritis (6), multiple sclerosis (7, 8), and Crohn''s disease (9). These observations suggest that blockade of IL-18 may be a useful human therapeutic modality (10).Despite functional divergence from the IL-1 cytokine family, IL-18 shares many similarities with IL-1. First, human IL-18 is synthesized as a biologically inactive 24-kDa precursor. Like IL-1β, IL-18 is activated and secreted following cleavage by caspase-1 (and possibly other proteases) that generates the mature 18-kDa polypeptide. Despite low sequence homology to IL-1β (17%), the three-dimensional structure of IL-18 closely resembles the IL-1β β-trefoil fold, as shown by a recent IL-18 NMR structure determination (1). The IL-1 and IL-18 receptors are also homologous; IL-18 binds either to the IL-18Rα chain alone or to the heterodimeric IL-18Rα/IL-18Rβ receptor complex. IL-18 binds to IL-18Rα with ∼20 nm affinity, but signaling occurs only upon formation of the high affinity (0.2 nm) IL-18Rα·IL-18·IL-18Rβ ternary complex (11, 12). Surface mutational analysis has identified two sites for IL-18 binding to IL-18Rα that are similar to those observed in the IL-1β·IL-1Rα binary complex (13) as well as one site important for binding to IL-18Rβ (1).In a recent study, a potent (0.2 nm) IL-18-neutralizing murine monoclonal antibody (mAb), 125-2H, inhibited binding of IL-18 to IL-18Rα alone but not the heterodimeric IL-18Rα/IL-18Rβ receptor complex, despite rendering the ternary complex with IL-18 non-functional (14). The structural basis for the unusual properties of 125-2H are unclear; the authors suggested that conformational changes in IL-18Rα occur upon formation of the IL-18Rα/IL-18Rβ receptor, thereby altering the interactions with 125-2H (14).To understand the intricate interactions between IL-18 and this antibody, we have determined the co-crystal structure of human IL-18 and the 125-2H antigen-binding fragment (Fab) at 1.5 Å resolution. This structure rationalizes epitope mapping data, based on human/murine IL-18 chimeras (14), in which the primary antigenic recognition loop is located near the COOH terminus. A secondary loop bolsters the interactions between IL-18 and several 125-2H complementarity-determining regions (CDRs). Comparison of this complex structure with that of the unbound 125-2H Fab (2.3 Å resolution) shows that 125-2H is preorganized for antigen binding. Last, we also have determined the 1.5 Å resolution crystal structure of the Fab fragment of a fully human mAb, ABT-325, that binds a distinct IL-18 epitope, as confirmed by biochemical studies. ABT-325 is entering clinical trials for a variety of autoimmune disease indications.     

Mycobacterium tuberculosis Rv3402c Enhances Mycobacterial Survival within Macrophages and Modulates the Host Pro-Inflammatory Cytokines Production via NF-Kappa B/ERK/p38 Signaling

Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis, a process which depends on an array of virulence factors to colonize and replicate within the host. The M. tuberculosis iron regulated open reading frame (ORF) rv3402c, encoding a conserved hypothetical protein, was shown to be up-regulated upon infection in both human and mice macrophages. To explore the function of this ORF, we heterologously expressed the rv3402c gene in the non-pathogenic fast-growing Mycobacterium smegmatis strain, and demonstrated that Rv3402c, a cell envelope-associated protein, was able to enhance the intracellular survival of recombinant M. smegmatis. Enhanced growth was not found to be the result of an increased resistance to intracellular stresses, as growth of the Rv3402c expressing strain was unaffected by iron depletion, H₂O₂ exposure, or acidic conditions. Colonization of macrophages by M. smegmatis expressing Rv3402c was associated with substantial cell death and significantly greater amount of TNF-α and IL-1β compared with controls. Rv3402c-induced TNF-α and IL-1β production was found to be mediated by NF-κB, ERK and p38 pathway in macrophages. In summary, our study suggests that Rv3402c delivered in a live M. smegmatis vehicle can modify the cytokines profile of macrophage, promote host cell death and enhance the persistence of mycobacterium within host cells.