Characterization of released polypeptides during an interferon-γ-dependent antibacterial response in airway epithelial cells.

When pathogenic bacteria breach the epithelial lining at mucosal surfaces, rapidly available innate immune mechanisms are critical to halt the infection. In the present study, we characterized the production of antibacterial polypeptides released by epithelial cells. IFN-γ, but neither TNF nor IL-1ß alone, induced release of antibacterial activity to a cell culture medium, causing a lytic appearance of killed bacteria as revealed by electron microscopy. Addition of the protein streptococcal inhibitor of complement, derived from Streptococcus pyogenes, known for its ability to neutralize antimicrobial polypeptides (AMPs), reduced the antibacterial activity of the medium. Characterization of the antibacterial incubation medium using mass spectrometric approaches and ELISAs, displayed presence of several classical AMPs, antibacterial chemokines, as well as complement factors and proteases that may interfere with bacterial killing. Many were constitutively produced, that is, being released by cells incubated in a medium alone. While a combination of IFN-γ and TNF did not increase bacterial killing, the presence of TNF boosted the amounts and detectable number of AMPs, including antibacterial chemokines. However, the methods applied in the study failed to single out certain AMPs as critical mediators, but rather demonstrate the broad range of molecules involved. Since many AMPs are highly amphiphatic in nature (i.e., cationic and hydrophobic), it is possible that difficulties in optimizing recovery present limitations in the context investigated. The findings demonstrate that epithelial cells have a constitutive production of AMPs and that IFN-γ is an important inducer of an antibacterial response in which is likely to be a critical part of the innate host defense against pathogenic bacteria at mucosal surfaces.

Peptidylarginine deiminases present in the airways during tobacco smoking and inflammation can citrullinate the host defense peptide LL-37, resulting in altered activities.

Bacterial colonization of the lower respiratory tract is frequently seen in chronic obstructive pulmonary disease (COPD), and may cause exacerbations leading to disease progression. Antimicrobial peptides comprise an important part of innate lung immunity, and not least the cathelicidin human cationic antimicrobial protein-18/LL-37. Peptidylarginine deiminases (PADIs) post-translationally modify proteins by converting cationic peptidylarginine residues to neutral peptidylcitrulline. An increased presence of PADI2 and citrullinated proteins was demonstrated in the lungs of smokers. In this study, preformed PADI4, stored in granulocytes and extracellularly in the lumina of bronchi, was found in lung tissue of individuals suffering from COPD. In vitro, recombinant human PADI2 and PADI4 both caused a time- and dose-dependent citrullination of LL-37. The citrullination resulted in impaired antibacterial activity against Staphylococcus aureus, Streptococcus pneumoniae, and nontypable Haemophilus influenzae, but less so against Pseudomonas aeruginosa. Using artificial lipid bilayers, we observed discrete differences when comparing the disrupting activity of native and citrullinated LL-37, suggesting that differences in cell wall composition are important during interactions with whole bacteria. Furthermore, citrullinated LL-37 showed higher chemotactic activity against mononuclear leukocytes than did native LL-37, but was less efficient at neutralizing lipolysaccharide, and also in converting apoptotic neutrophils into a state of secondary necrosis. In addition, citrullinated LL-37 was more prone to degradation by proteases, whereas the V8 endopetidase of S. aureus cleaved the modified peptide at additional sites, compared with native LL-37. Together, these findings demonstrate novel mechanisms whereby the inflammation-dependent deiminases PADI2 and PADI4 can alter the activites of antibacterial polypeptides, affecting the course of inflammatory disorders such as COPD.

Streptococcus pneumoniae induces expression of the antibacterial CXC chemokine MIG/CXCL9 via MyD88-dependent signaling in a murine model of airway infection.

MIG/CXCL9 belongs to the CXC family of chemokines and participates in the regulation of leukocyte-trafficking and angiogenesis. Certain chemokines, including human MIG/CXCL9, exert strong antibacterial activity in vitro, although the importance of this property in vivo is unknown. In the present study, we investigated the expression and a possible role for MIG/CXCL9 in host defense during mucosal airway infection caused by Streptococcus pneumoniae in vivo. We found that intranasal challenge of C57BL/6 wild-type mice with pneumococci elicited production of high levels of MIG/CXCL9 in the lungs via the MyD88-dependent signaling pathway. Whereas both human and murine MIG/CXCL9 showed efficient killing of S. pneumoniae in vitro, MIG/CXCL9 knock-out mice were not more susceptible to pneumococcal infection. Our data demonstrate that, in vivo this chemokine probably has a redundant role, acting together with other antibacterial peptides and chemokines, in innate and adaptive host defense mechanisms against pneumococcal infections.

SufA of the opportunistic pathogen finegoldia magna modulates actions of the antibacterial chemokine MIG/CXCL9, promoting bacterial survival during epithelial inflammation.

The anaerobic bacterium Finegoldia magna is part of the human commensal microbiota, but is also an important opportunistic pathogen. This bacterium expresses a subtilisin-like serine proteinase, SufA, which partially degrade the antibacterial chemokine MIG/CXCL9. Here, we show that MIG/CXCL9 is produced by human keratinocytes in response to inflammatory stimuli. In contrast to the virulent human pathogen Streptococcus pyogenes, the presence of F. magna had no enhancing effect on the MIG/CXCL9 expression by keratinocytes, suggesting poor detection of the latter by pathogen-recognition receptors. When MIG/CXCL9 was exposed to SufA-expressing F. magna, the molecule was processed into several smaller fragments. Analysis by mass spectrometry showed that SufA cleaves MIG/CXCL9 at several sites in the COOH-terminal region of the molecule. At equimolar concentrations, SufA-generated MIG/CXCL9 fragments were not bactericidal against F. magna, but retained their ability to kill S. pyogenes. Moreover, the SufA-generated MIG/CXCL9 fragments were capable of activating the angiostasis-mediating CXCR3 receptor, which is expressed on endothelial cells, in an order of magnitude similar to that of intact MIG/CXCL9. F. magna expresses a surface protein called FAF that is released from the bacterial surface by SufA. Soluble FAF was found to bind and inactivate the antibacterial activity of MIG/CXCL9, thereby further potentially promoting the survival of F. magna. The findings suggest that SufA modulation of the inflammatory response could be a mechanism playing an important role in creating an ecologic niche for F. magna, decreasing antibacterial activity and suppressing angiogenesis, thus providing advantage in survival for this anaerobic opportunist compared with competing pathogens during inflammation.

Antibacterial chemokines--actors in both innate and adaptive immunity.

Several antibacterial proteins and peptides of the human innate immune system have additional roles in the regulation of adaptive immune responses. Among peptides with innate and adaptive immune functions are chemokines, a family of structurally related peptides with conserved amino-terminal motifs. Chemokines regulate leukocyte trafficking during both health and disease. In recent years, some chemokines have been shown to exert direct antibacterial activity. On the other hand, several granulebound antibacterial proteins of granulocytes, and epithelium-expressed antibacterial polypeptides, possess chemotactic activity and stimulate cells of the adaptive immune system. It is likely that during evolution, some antimicrobial peptides and proteins of innate immunity have diverged to coordinate the actions of the innate immune system and the evolutionary younger, adaptive immunity. This review aims to describe antibacterial chemokines and antibacterial peptides possessing chemotactic activity, biologic properties that link innate and adaptive immunity.

M1 protein of Streptococcus pyogenes increases production of the antibacterial CXC chemokine MIG/CXCL9 in pharyngeal epithelial cells.

Streptococcus pyogenes adheres to epithelial cells of the human pharynx where it can cause pharyngitis. To counteract infection, inflamed epithelium produces peptide antibiotics, among them the CXC chemokine MIG/CXCL9. M protein is both a surface-associated and released virulence factor of S. pyogenes. Here, we show that soluble M1 protein enhances MIG gene expression and synthesis in IFN-gamma stimulated epithelial cells. M1 protein was recognized both by resting and IFN-gamma activated pharyngeal epithelial cells as detected by activation of the transcription factor NF-kappaB. Furthermore, pharmacological inhibition of NF-kappaB, decreased MIG synthesis in IFN-gamma activated cells, demonstrating a key role for NF-kappaB in mediating the enhanced response. Microarrays were used to investigate expression of recognized antimicrobial peptides in pharyngeal epithelial cells after stimulation with a combination of IFN-gamma and M1 protein. Amongst the most up-regulated and expressed genes, were several antibacterial CC and CXC chemokines. To investigate an in vivo context, pharyngeal mucosa was stimulated in vitro and MIG could be detected by immunohistochemistry in epithelial cells. The results show that epithelial cells can recognize solubilized M1 protein and intact S. pyogenes, thereby modulating an antibacterial innate host response that may have bearing on the outcome of streptococcal pharyngitis.

The proinflammatory CXC-chemokines GRO-alpha/CXCL1 and MIG/CXCL9 are concomitantly expressed in ulcerative colitis and decrease during treatment with topical corticosteroids.

BACKGROUND: Ulcerative colitis is characterized by relapsing mucosal inflammation where the lesions include tissue-damaging granulocytes. In addition, T cells and natural killer (NK) cells play important pathophysiologic roles. Chemokines are a large family of peptides that play key roles in the regulation of inflammation. The CXC-chemokines, growth-related oncogene (GRO)-alpha/CXCL1 and interleukin (IL)-8/CXCL8, both recruit neutrophils and possess mitogenic properties, whereas the interferon-dependent CXC-chemokines monokine induced by gamma-interferon (MIG)/CXCL9, interferon-gamma inducible protein of 10 kD/CXCL10, and IFN-inducible T cell alpha chemoattractant/CXCL11 recruit and activate T cells and NK cells. MATERIALS AND METHODS: The expression of CXC-chemokines was studied in eight controls and in 11 patients suffering from ulcerative colitis in the distal part of the colon, before and during topical treatment with corticosteroids. Perfusates (obtained before, after 7 days, and after 28 days of treatment) and pinch biopsies (obtained before and after 28 days of treatment) were collected by colonoscopy. The rectal release of GRO-alpha and MIG was determined by enzyme-linked immunosorbent assay (ELISA), and tissue expression of the chemokines was detected in colonic tissue by immunohistochemistry. RESULTS: In perfusates, high levels of GRO-alpha, IL-8, and MIG were detected compared with controls (p=0.02, 0.005, and p=0.03, respectively). During treatment with corticosteroids, both GRO-alpha and MIG decreased. In clinical nonresponders, characterized by sustained inflammation, the levels of GRO-alpha and MIG remained elevated. Both epithelial cells and granulocytes, present in the submucosa, expressed GRO-alpha and MIG as detected by immunohistochemistry. CONCLUSIONS: CXC-chemokines are likely to be important in the pathophysiology of ulcerative colitis and may become targets for novel treatment strategies. In addition, GRO-alpha may serve as a marker of disease activity.

The CXC chemokine MIG/CXCL9 is important in innate immunity against Streptococcus pyogenes.

Pharyngitis caused by Streptococcus pyogenes is one of the most common bacterial infections in humans and is also a starting point for invasive S. pyogenes infection. Here, we describe that tonsil fluid from patients with streptococcal pharyngitis contains high amounts of the interferon (IFN)-dependent CXC chemokine known as monokine induced by IFN- gamma (MIG)/CXCL9. Also in vitro, inflamed pharyngeal epithelium produced large amounts of MIG/CXCL9 in the presence of bacteria. The CXC chemokines MIG/CXCL9, IFN-inducible protein-10/CXCL10, and IFN-inducible T cell alpha -chemoattractant/CXCL11 all showed antibacterial activity against S. pyogenes, and inhibition of MIG/CXCL9 expression reduced the antibacterial activity at the surface of inflamed pharyngeal cells. S. pyogenes of the clinically important M1 serotype secrets the protein streptococcal inhibitor of complement (SIC), which inhibited the antibacterial activity of the chemokines. As exemplified by S. pyogenes pharyngitis, the data identify a novel innate defense mechanism against invasive bacteria on epithelial surfaces.