Interleukin-22 regulates the complement system to promote resistance against pathobionts after pathogen-induced intestinal damage.

Pathobionts play a critical role in disease development, but the immune mechanisms against pathobionts remain poorly understood. Here, we report a critical role for interleukin-22 (IL-22) in systemic protection against bacterial pathobionts that translocate into the circulation after infection with the pathogen Clostridium difficile. Infection with C. difficile induced IL-22, and infected Il22(-/-) mice harbored high numbers of pathobionts in extraintestinal organs despite comparable pathogen load and intestinal damage in mutant and wild-type mice. Pathobionts exhibited increased resistant against complement-mediated phagocytosis, and their intravenous administration resulted in high animal mortality. Selective removal of translocated commensals rescued Il22(-/-) mice, and IL-22 administration enhanced the elimination of pathobionts. Mechanistically, IL-22 augmented bacterial phagocytosis by increasing the expression and bacterial binding of complement C3. Our study demonstrates an unexpected role for IL-22 in controlling the elimination of pathobionts that enter the systemic circulation through the regulation of the complement system.

Type 3 hypersensitivity in COVID-19 vasculitis.

Coronavirus Disease 2019 (COVID-19) is an ongoing public health emergency and new knowledge about its immunopathogenic mechanisms is deemed necessary in the attempt to reduce the death burden, globally. For the first time in worldwide literature, we provide scientific evidence that in COVID-19 vasculitis a life-threatening escalation from type 2 T-helper immune response (humoral immunity) to type 3 hypersensitivity (immune complex disease) takes place. The subsequent deposition of immune complexes inside the vascular walls is supposed to induce a severe inflammatory state and a cytokine release syndrome, whose interleukin-6 is the key myokine, from the smooth muscle cells of blood vessels.

Contribution of acute-phase reaction proteins to the diagnosis and treatment of 2019 novel coronavirus disease (COVID-19).

The emergence of 2019 novel coronavirus disease (COVID-19) is currently a global concern. In this study, our goal was to explore the changing expression levels of acute-phase reaction proteins (APRPs) in the serum of COVID-19 patients and to elucidate the immunological characteristics of COVID-19. In the study design, we recruited 72 COVID-19 patients, including 22 cases of mild degree, 38 cases of moderate degree and 12 cases of severe degree. We also recruited 20 patients with community-acquired pneumonia (CAP) and 20 normal control subjects as a comparison. Fasting venous blood was taken to detect the content of complement 3 (C3), complement 4 (C4), C-reactive protein (CRP), serum amyloid A (SAA) and prealbumin (PA). When compared the COVID-19 group with the CAP and normal control groups, respectively, the mean value of CRP and SAA in the COVID-19 group (including mild, moderate and severe patients) had increased significantly (P < 0.01), whereas the mean values of C3, C4 and PA decreased (P < 0.01). For the asymptomatic or mild symptomatic patients with COVID-19, the actual aggravation of disease may be more advanced than the clinical appearances. Meanwhile, the statistical analyses indicated that the development of COVID-19 brought about a significant increase in the content of CRP and SAA (P < 0.01), and a decline in the content of C3, C4 and PA (P < 0.01). These findings suggested that the changes in the level of APRPs could be used as indicators to identify the degree and progression of COVID-19, and the significant changes might demonstrate the aggravation of disease. This study provided a new approach to improve the clinical management plan and prognosis of COVID-19.

The relationship between complement C3 expression and the MUC5B genotype in pulmonary fibrosis.

The common gain-of-function MUC5B promoter variant ( rs35705950 ) is the strongest risk factor for the development of idiopathic pulmonary fibrosis (IPF). While the role of complement in IPF is controversial, both MUC5B and the complement system play a role in lung host defense. The aim of this study was to evaluate the relationship between complement component 3 (C3) and MUC5B in patients with IPF and in bleomycin-induced lung injury in mice. To do this, we evaluated C3 gene expression in whole lung tissue from 300 subjects with IPF and 175 healthy controls. Expression of C3 was higher in IPF than healthy controls {1.40-fold increase [95% confidence interval (CI) 1.31-1.50]; P < 0.0001} and even greater among IPF subjects with the highest-risk IPF MUC5B promoter genotype [TT vs. GG = 1.59-fold (95% CI 1.15-2.20); P < 0.05; TT vs. GT = 1.66-fold (95% CI 1.20-2.30); P < 0.05]. Among subjects with IPF, C3 expression was significantly higher in the lung tissue without microscopic honeycombing than in the lung tissue with microscopic honeycombing [1.40-fold increase (95% CI 1.23- 1.59); P < 0.01]. In mice, while bleomycin exposure increased Muc5b protein expression, C3-deficient mice were protected from bleomycin-induced lung injury. In aggregate, our findings indicate that the MUC5B promoter variant is associated with higher C3 expression and suggest that the complement system may contribute to the pathogenesis of IPF.

Complement Component 3 Is Regulated by TWIST1 and Mediates Epithelial-Mesenchymal Transition.

We have previously shown that complement component 3 (C3) is secreted by malignant epithelial cells. To understand the mechanism of upregulation of C3 expression in tumor cells, we studied the C3 promoter and identified that twist basic helix-loop-helix transcription factor 1 (TWIST1) binds to the C3 promoter and enhances its expression. Because TWIST1 mediates epithelial-mesenchymal transition (EMT), we studied the effect of C3 on EMT and found that C3 decreased E-cadherin expression on cancer cells and promoted EMT. We showed that C3-induced reduction in E-cadherin expression in ovarian cancer cells was mediated by C3a and is Krüppel-like factor 5 dependent. We investigated the association between TWIST1 and C3 in malignant tumors and in murine embryos. TWIST1 and C3 colocalized at the invasive tumor edges, and in the neural crest and limb buds of mouse embryos. Our results identified TWIST1 as a transcription factor that regulates C3 expression during pathologic and physiologic EMT.

HK2 Proximal Tubule Epithelial Cells Synthesize and Secrete Plasma Proteins Predominantly Through the Apical Surface.

Renal proximal tubule epithelial cells (PTECs) are known to reabsorb salts and small plasma proteins filtered through Bowman's capsule. Following acute kidney injury, PTECs assume some characteristics of hepatocytes in producing various plasma proteins. We now demonstrate that even at a resting state, a PTEC cell line, HK2 expresses mRNAs for and synthesizes and secretes plasma proteins in a complex with complement C3, an α2 -macroglobulin family chaperone, including albumin, transferrin, α1 -antitrypsin, α1 -antichymotrypsin, α2 -HS-glycoprotein, ceruloplasmin, haptoglobin, C1-inhibitor, secreted phosphoprotein-24, and insulin-like growth factor-1. When grown on transwell inserts, HK2 cells predominantly secrete (∼90%) plasma proteins into the apical side and a smaller fraction into the basolateral side as determined by ELISA assays. When cultured in the presence of exogenous cytokines such as IL1ß, IL6, TNFα, BMP2, or TGFß1, HK2 cell mRNA expressions for plasma proteins were variably affected whereas basolateral secretions were elevated to or in excess of those of the apical level. In addition, HK2 cells produce proTGFß1 with its intact N-terminal latency associated peptide and latent-TGF-ß-binding proteins. The complex cannot be dissociated under conditions of SDS, heating, and electrophoresis. Moreover, HK2 cells maintain their ability to quickly uptake exogenously added serum proteins from the culture medium, as if they are recognized differently by the endocytic receptors. These results provide new insight into the hepatization of PTECs. In addition to their unique uptake of plasma proteins and salts from the filtrate, they are a source of urinary proteins under normal conditions as wells as in chronic and acute kidney diseases. J. Cell. Biochem. 118: 924-933, 2017. © 2016 Wiley Periodicals, Inc.

Unbiased expression mapping identifies a link between the complement and cholinergic systems in the rat central nervous system.

The complement system is activated in a wide spectrum of CNS diseases and is suggested to play a role in degenerative phenomena such as elimination of synaptic terminals. Still, little is known of mechanisms regulating complement activation in the CNS. Loss of synaptic terminals in the spinal cord after an experimental nerve injury is increased in the inbred DA strain compared with the PVG strain and is associated with expression of the upstream complement components C1q and C3, in the absence of membrane attack complex activation and neutrophil infiltration. To further dissect pathways regulating complement expression, we performed genome-wide expression profiling and linkage analysis in a large F2(DA × PVG) intercross, which identified quantitative trait loci regulating expression of C1qa, C1qb, C3, and C9. Unlike C1qa, C1qb, and C9, which all displayed distinct coregulation with different cis-regulated C-type lectins, C3 was regulated in a coexpression network immediately downstream of butyrylcholinesterase. Butyrylcholinesterase hydrolyses acetylcholine, which exerts immunoregulatory effects partly through TNF-α pathways. Accordingly, increased C3, but not C1q, expression was demonstrated in rat and mouse glia following TNF-α stimulation, which was abrogated in a dose-dependent manner by acetylcholine. These findings demonstrate new pathways regulating CNS complement expression using unbiased mapping in an experimental in vivo system. A direct link between cholinergic activity and complement activation is supported by in vitro experiments. The identification of distinct pathways subjected to regulation by naturally occurring genetic variability is of relevance for the understanding of disease mechanisms in neurologic conditions characterized by neuronal injury and complement activation.

Enrichment of immunoregulatory proteins in the biomolecular corona of nanoparticles within human respiratory tract lining fluid.

When inhaled nanoparticles deposit in the lungs, they transit through respiratory tract lining fluid (RTLF) acquiring a biomolecular corona reflecting the interaction of the RTLF with the nanomaterial surface. Label-free snapshot proteomics was used to generate semi-quantitative profiles of corona proteins formed around silica (SiO2) and poly(vinyl) acetate (PVAc) nanoparticles in RTLF, the latter employed as an archetype drug delivery vehicle. The evolved PVAc corona was significantly enriched compared to that observed on SiO2 nanoparticles (698 vs. 429 proteins identified); however both coronas contained a substantial contribution from innate immunity proteins, including surfactant protein A, napsin A and complement (C1q and C3) proteins. Functional protein classification supports the hypothesis that corona formation in RTLF constitutes opsonisation, preparing particles for phagocytosis and clearance from the lungs. These data highlight how an understanding of the evolved corona is necessary for the design of inhaled nanomedicines with acceptable safety and tailored clearance profiles. FROM THE CLINICAL EDITOR: Inhaled nanoparticles often acquire a layer of protein corona while they go through the respiratory tract. Here, the authors investigated the identity of these proteins. The proper identification would improve the understanding of the use of inhaled nanoparticles in future therapeutics.

Hepatitis C virus impairs natural killer cell-mediated augmentation of complement synthesis.

UNLABELLED: Natural killer (NK) cells and the complement system play critical roles in the first line of defense against pathogens. The synthesis of complement components C4 and C3 is transcriptionally downregulated by hepatitis C virus (HCV) core and NS5A proteins, and this negative regulation is apparent in chronically HCV-infected patients. In this study, we have examined the potential contribution of an NK cell line as a model in regulating complement synthesis. Coculture of NK cells (NK3.3) with human hepatoma cells (Huh7.5) expressing HCV core or NS5A protein led to a significant increase in C4 and C3 complement synthesis via enhanced specific transcription factors. Reestablishment of complement protein expression was found to be mediated by direct interaction between NKG2D on NK cells and the hepatocyte protein major histocompatibility complex class I-related chains A and B (MICA/B) and not to be associated with specific cytokine signaling events. On the other hand, C4 and C3 synthesis remained impaired in a coculture of NK cells and Huh7.5 cells infected with cell culture-grown HCV. The association between these two cell types through NKG2D and MICA/B was examined further, with MICA/B expression in HCV-infected hepatocytes found to remain inhibited during coculture. Further experiments revealed that the HCV NS2 and NS5B proteins are responsible for the HCV-associated decrease in MICA/B. These results suggest that HCV disables a key receptor ligand in infected hepatoma cells, thereby inhibiting the ability of infected cells to respond to stimuli from NK cells to positively regulate complement synthesis. IMPORTANCE: The complement system contributes to the protection of the host from virus infection. However, the involvement of complement in viral hepatitis has not been well documented. Whether NK cells affect complement component expression in HCV-infected hepatocytes remains unknown. Here, we have shown how HCV subverts the ability of NK cells to positively mediate complement protein expression.

Deletion of Crry and DAF on murine platelets stimulates thrombopoiesis and increases factor H-dependent resistance of peripheral platelets to complement attack.

Complement receptor 1-related gene/protein y (Crry) and decay-accelerating factor (DAF) are two murine membrane C3 complement regulators with overlapping functions. Crry deletion is embryonically lethal whereas DAF-deficient mice are generally healthy. Crry(-/-)DAF(-/-) mice were viable on a C3(-/-) background, but platelets from such mice were rapidly destroyed when transfused into C3-sufficient mice. In this study, we used the cre-lox system to delete platelet Crry in DAF(-/-) mice and studied Crry/DAF-deficient platelet development in vivo. Rather than displaying thrombocytopenia, Pf4-Cre(+)-Crry(flox/flox) mice had normal platelet counts and their peripheral platelets were resistant to complement attack. However, chimera mice generated with Pf4-Cre(+)-Crry(flox/flox) bone marrows showed platelets from C3(-/-) but not C3(+/+) recipients to be sensitive to complement activation, suggesting that circulating platelets in Pf4-Cre(+)-Crry(flox/flox) mice were naturally selected in a complement-sufficient environment. Notably, Pf4-Cre(+)-Crry(flox/flox) mouse platelets became complement susceptible when factor H function was blocked. Examination of Pf4-Cre(+)-Crry(flox/flox) mouse bone marrows revealed exceedingly active thrombopoiesis. Thus, under in vivo conditions, Crry/DAF deficiency on platelets led to abnormal platelet turnover, but peripheral platelet count was compensated for by increased thrombopoiesis. Selective survival of Crry/DAF-deficient platelets aided by factor H protection and compensatory thrombopoiesis demonstrates the cooperation between membrane and fluid phase complement inhibitors and the body's ability to adaptively respond to complement regulator deficiencies.

Human skin mast cells express complement factors C3 and C5.

We examine whether complement factor C3 or C5 is synthesized by human skin-derived mast cells and whether their synthesis is regulated by cytokines. C3 and C5 mRNAs were assessed by RT-PCR, and proteins by flow cytometry, confocal microscopy, Western blotting, and ELISA. C3 and C5 mRNAs were each expressed, and baseline protein levels/10(6) cultured mast cells were 0.9 and 0.8 ng, respectively, and located in the cytoplasm outside of secretory granules. C3 accumulated in mast cell culture medium over time and by 3 d reached a concentration of 9.4 ± 8.0 ng/ml, whereas C5 levels were not detectable (<0.15 ng/ml). Three-day incubations of mast cells with IL-1α, IL-1ß, IL-17, IFN-γ, IL-6, or anti-FcεRI did not affect C3 protein levels in culture medium, whereas incubations with PMA, TNF-α, IL-13, or IL-4 enhanced levels of C3 1.7- to 3.3-fold. In contrast with C3, levels of C5 remained undetectable. Importantly, treatment with TNF-α together with either IL-4 or IL-13 synergistically enhanced C3 (but not C5) production in culture medium by 9.8- or 7.1-fold, respectively. This synergy was blocked by attenuating the TNF-α pathway with neutralizing anti-TNF-α Ab, soluble TNFR, or an inhibitor of NF-κB, or by attenuating the IL-4/13 pathway with Jak family or Erk antagonists. Inhibitors of PI3K, Jnk, and p38 MAPK did not affect this synergy. Thus, human mast cells can produce and secrete C3, whereas ß-tryptase can act on C3 to generate C3a and C3b, raising the likelihood that mast cells engage complement to modulate immunity and inflammation in vivo.

Determination of reference intervals for serum complement C3 and C4 levels in Chinese Han ethnic males.

BACKGROUND: Clinical laboratory reference intervals (RIs) for serum complement C3 and C4 levels have been established in many countries but there is a lack of published data regarding normal RIs in Chinese population. We attempted to establish RIs for serum complement C3 and C4 levels in Chinese Han ethnic males. METHODS: A total of 1,234 healthy male subjects, aged 20 - 69 years, were collected from the Fangchenggang Area Male Health and Examination Survey (FAMHES). Serum complement C3 and C4 levels were measured by immunoturbidimetry. The two-sided 95-percentile RIs were calculated using parametric statistical methods. RESULTS: Serum C3 values showed normal distribution and C4 were log-normal distributed. The two-sided 95% RIs (mean +/- 2 SD) for serum C3 and C4 were 0.656 - 1.52 g/L and 0.181 - 0.561 g/L, respectively. Body Mass Index (BMI) had a significant positive association with C3 (r = 0.342) and C4 (r = 0.258), and age had a significant positive association with C4 (r = 0.117). No significant difference was found either between smoking groups or drinking groups. A significant increase with BMI was found both for C3 (p < 0.001) and C4 (p < 0.001). BMI-specific RIs were also calculated. CONCLUSIONS: The RIs for serum C3 and C4 show a slight deviation compared to previously reported reference levels. BMI-specific reference values should be implemented in clinical laboratories.

Modified low density lipoprotein stimulates complement C3 expression and secretion via liver X receptor and Toll-like receptor 4 activation in human macrophages.

Complement C3 is a pivotal component of three cascades of complement activation. C3 is expressed in human atherosclerotic lesions and is involved in atherogenesis. However, the mechanism of C3 accumulation in atherosclerotic lesions is not well elucidated. We show that acetylated low density lipoprotein and oxidized low density lipoprotein (oxLDL) increase C3 gene expression and protein secretion by human macrophages. Modified LDL (mLDL)-mediated activation of C3 expression mainly depends on liver X receptor (LXR) and partly on Toll-like receptor 4 (TLR4), whereas C3 secretion is increased due to TLR4 activation by mLDL. LXR agonist TO901317 stimulates C3 gene expression in human monocyte-macrophage cells but not in human hepatoma (HepG2) cells. We find LXR-responsive element inside of the promoter region of the human C3 gene, which binds to LXRß in macrophages but not in HepG2 cells. We show that C3 expression and secretion is decreased in IL-4-treated (M2) and increased in IFNγ/LPS-stimulated (M1) human macrophages as compared with resting macrophages. LXR agonist TO901317 potentiates LPS-induced C3 gene expression and protein secretion in macrophages, whereas oxLDL differently modulates LPS-mediated regulation of C3 in M1 or M2 macrophages. Treatment of human macrophages with anaphylatoxin C3a results in stimulation of C3 transcription and secretion as well as increased oxLDL accumulation and augmented oxLDL-mediated up-regulation of the C3 gene. These data provide a novel mechanism of C3 gene regulation in macrophages and suggest new aspects of cross-talk between mLDL, C3, C3a, and TLR4 during development of atherosclerotic lesions.

A novel mutation in the complement component 3 gene in a patient with selective IgA deficiency.

PURPOSE: Immunological and molecular evaluation of a patient presenting with recurrent infections caused by Streptococcus pneumoniae and low complement component 3 (C3) levels. METHODS: Immunological evaluation included complement components and immunoglobulin level quantification as well as number and function of T cells, B cells and neutrophils. Serotype-specific immunoglobulin G antibodies against S. pneumoniae capsular polysaccharides were quantified by ELISA in serum samples before and after vaccination with unconjugated polysaccharide vaccine. For the molecular analysis, genomic DNA from the patient and parents were isolated and all exons as well as exon-intron boundaries of the C3 gene were sequenced by Sanger sequencing. RESULTS: A 16-year-old male, born to consanguineous parents, presented with recurrent episodes of pneumonia caused by S. pneumoniae and bronchiectasis. The patient showed severely reduced C3 and immunoglobulin A levels, while the parents showed moderately reduced levels of C3. Mutational analysis revealed a novel, homozygous missense mutation in the C3 gene (c. C4554G, p. Cys1518Trp), substituting a highly conserved amino acid in the C345C domain of C3 and interrupting one of its disulfide bonds. Both parents were found to be carriers of the affected allele. Vaccination against S. pneumoniae resulted in considerable clinical improvement. CONCLUSIONS: We report a novel homozygous mutation in the C3 gene in a patient with concomitant selective IgA deficiency who presented with a marked clinical improvement after vaccination against S. pneumoniae. This observation underlines the notion that vaccination against this microorganism is an important strategy for treatment of PID patients, particularly those presenting with increased susceptibility to infections caused by this agent.

Profiling the potential tumor markers of pancreatic ductal adenocarcinoma using 2D-DIGE and MALDI-TOF-MS: up-regulation of Complement C3 and alpha-2-HS-glycoprotein.

BACKGROUND/AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with an increasing incidence worldwide. Due to lack of early diagnosis and poor prognosis, it is rather critical to improve the early diagnosis of PDAC. A comparative proteomic method was used to analyze serum proteins to find a new potential specific marker. METHODS: Comparative analysis of the pancreatic peripheral blood protein profiling from 40 pancreatic cancer patients, 10 pancreatic benign tumor patients, 10 chronic pancreatitis patients and 40 cancer-free controls. The samples were carried out by 2D-differential gel electrophoresis (2D-DIGE) and differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Two up-regulated proteins were further validation by real time RT-PCR, Western blot analysis and Immunohistochemistry (IHC). RESULTS: We identified fourteen differently expressed proteins in PDAC group compared with cancer-free control group, including 9 up-regulation and 5 down-regulation proteins. Increased Complement C3 and alpha-2-HS-glycoprotein (AHSG) were further confirmed by real time RT-PCR, Western blot analysis and IHC. The expressions of Complement C3 and AHSG were higher in PDAC than that in other groups. CONCLUSIONS: These results suggest that Complement C3 and AHSG might be the potential tumor markers in PDAC screening and diagnosis. The finding of inflammation mediated factor Complement C3 revealed that inflammation might be closely related with the occurrence and development process of PDAC.

Complement factor C5 deficiency significantly delays the progression of biliary fibrosis in bile duct-ligated mice.

Fibrogenesis represents the universal response of the liver to chronic liver injury. Complement factor C5 has been linked to fibrosis in murine toxic liver injury and human chronic hepatitis C. C5 may also play a central role in chronic cholestatic disorders, since the BA receptor FXR has been characterized as an activator of the C3 gene. We aimed to investigate, whether C5 deficiency is able to prevent biliary fibrosis in the mouse bile-duct-ligation model. BDL for 1-4 weeks was performed in either Hc(0)/Hc(0) mice (deficient for C5) or WT controls. BA levels were measured by RIA. Histological examination included H&E, sirius-red and immunohistochemistry. mRNA expression was quantified by RT-PCR. Protein expression levels were determined by Western blotting or ELISA. Enzymatic MMP-activity was analysed by zymography. One week BDL leads to fibrosis in WT (F2.0 ± 0), while it is almost absent in Hc(0)/Hc(0) mice (F0.5 ± 0.5). No differences in fibrosis can be detected at week-4. Together with delayed fibrogenesis at week-1, fibrotic markers are decreased in Hc(0)/Hc(0) mice. Expression of the inflammatory cytokine TNF-α is decreased in Hc(0)/Hc(0) mice. In parallel C5 deficiency leads to an attenuated peribiliary infiltration of CD45(+) cells in fibrotic areas together with decreased MMP-9 expression and gelatinase activity. The present study proves a functional role of C5 during biliary fibrogenesis. C5 deficiency leads to attenuated inflammation and normalized MMP-9 activity concomitantly with a significant reduction of fibrosis. C5 appears to be an attractive target for future therapeutic intervention in chronic cholestatic liver disease.

Suppression of cytokine-mediated complement factor gene expression through selective activation of the Ah receptor with 3',4'-dimethoxy-α-naphthoflavone.

We have characterized previously a class of aryl hydrocarbon receptor (AHR) ligand termed selective AHR modulators (SAhRMs). SAhRMs exhibit anti-inflammatory properties, including suppression of cytokine-mediated acute phase genes (e.g., Saa1), through dissociation of non-dioxin-response element (DRE) AHR activity from DRE-dependent xenobiotic gene expression. The partial AHR agonist α-naphthoflavone (αNF) mediates the suppressive, non-DRE dependent effects on SAA1 expression and partial DRE-mediated CYP1A1 induction. These observations suggest that αNF may be structurally modified to a derivative exhibiting only SAhRM activity. A screen of αNF derivatives identifies 3',4'-dimethoxy-αNF (DiMNF) as a candidate SAhRM. Competitive ligand binding validates DiMNF as an AHR ligand, and DRE-dependent reporter assays with quantitative mRNA analysis of AHR target genes reveal minimal agonist activity associated with AHR binding. Consistent with loss of agonist activity, DiMNF fails to promote AHR binding to DRE probes as determined through electromobility shift assay. Importantly, mRNA analysis indicates that DiMNF retains the suppressive capacity of αNF regarding cytokine-mediated SAA1 expression in Huh7 cells. Interestingly, predictive docking modeling suggests that DiMNF adopts a unique orientation within the AHR ligand binding pocket relative to αNF and may facilitate the rational design of additional SAhRMs. Microarray studies with a non-DRE binding but otherwise functional AHR mutant identified complement factor C3 as a potential SAhRM target. We confirmed this observation in Huh7 cells using 10 µM DiMNF, which significantly repressed C3 mRNA and protein. These data expand the classes of AHR ligands exerting DRE-independent anti-inflammatory SAhRM activity, suggesting SAhRMs may have application in the amelioration of inflammatory disorders.

Essential role of factor B of the alternative complement pathway in complement activation and opsonophagocytosis during acute pneumococcal otitis media in mice.

We recently reported that the complement system plays a pivotal role in innate immune defense against Streptococcus pneumoniae during acute otitis media (OM) in mice. The current study was designed to determine which of the complement pathways are activated during acute pneumococcal OM and whether components of complement are expressed in the middle ear epithelium. Gene expression was determined by quantitative PCR, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining. We found that S. pneumoniae induced increased gene expression of factor B of the alternative complement pathway and C3 in mouse middle ear epithelium. Activation of factor B and C3 in the middle ear lavage fluids was significantly greater than in simultaneously obtained serum samples as determined by Western blotting. Using mice deficient in complement C1qa, factor B, and factor B/C2, we found that complement C3 activation and opsonophagocytosis of S. pneumoniae were greatly attenuated in factor B- and factor B/C2-deficient mice. These findings support the concept that local complement activation is an important host innate immune response and that activation of the alternative complement pathway represents one of the innate immune defense mechanisms against pneumococcal infection during the early stage of acute OM.

The Sbi protein is a multifunctional immune evasion factor of Staphylococcus aureus.

The second immunoglobulin-binding protein (Sbi) of Staphylococcus aureus has two N-terminal domains that bind the Fc region of IgG in a fashion similar to that of protein A and two domains that can bind to the complement protein C3 and promote its futile consumption in the fluid phase. It has been proposed that Sbi helps bacteria to avoid innate immune defenses. By comparing a mutant defective in Sbi with mutants defective in protein A, clumping factor A, iron-regulated surface determinant H, and capsular polysaccharide, it was shown that Sbi is indeed an immune evasion factor that promotes bacterial survival in whole human blood and the avoidance of neutrophil-mediated opsonophagocytosis. Sbi is present in the culture supernatant and is also associated with the cell envelope. S. aureus strains that expressed truncates of Sbi lacking N-terminal domains D1 and D2 (D1D2) or D3 and D4 (D3D4) or a C-terminal truncate that was no longer retained in the cell envelope were analyzed. Both the secreted and envelope-associated forms of Sbi contributed to immune evasion. The IgG-binding domains contributed only when Sbi was attached to the cell, while only the secreted C3-binding domains were biologically active.