Moraxella catarrhalis Activates Murine Macrophages through Multiple Toll Like Receptors and Has Reduced Clearance in Lungs from TLR4 Mutant Mice

Ferdaus Hassan,Xin-Xing Gu,Wenhong Zhang,Dabin Ren,Tod J Merkel,Stefan Bereswill

doi:10.1371/journal.pone.0037610

Abstract

Moraxella catarrhalis is a Gram negative bacterium and a leading causative agent of otitis media (OM) in children. Several recent reports have provided strong evidence for an association between toll like receptors and OM. It has been found that both Streptococcus pneumoniae and nontypeable Haemophilus influenzae activate host protective immune responses through toll like receptors (TLRs), however, the precise mechanism by which Moraxella catarrhalis initiates the host immune response is currently unknown. In this report, using murine macrophages generated from a series of knock-out mice, we have demonstrated that M. catarrhalis lipooligosaccharide (LOS) and either heat killed or live bacteria are recognized by one or more TLRs. LOS activates the host immune response through a membrane bound CD14-TLR4 complex, while both heat killed and live M.cat require recognition by multiple toll like receptors such as TLR2, TLR4 and TLR9 without the requirement of CD14. We have also shown that M.cat stimuli are capable of triggering the host innate immune response by both MyD88- and TRIF- dependent signaling pathways. We further showed that M.cat induced activation of mitogen activated protein kinase (MAPK) is essential in order to achieve optimal secretion of pro-inflammatory cytokine TNF-α. We finally showed that TLR4 mutant C3H/HeJ mice produce significantly lower levels of pro-inflammatory cytokines TNF-α and IL-6 in vivo, An increased bacterial loads at 12 and 24 hours (P<0.001) in their lungs upon challenge with live M.cat in an aerosol chamber compared to wild-type (WT) control mice. These data suggest that TLRs are crucial for an effective innate immune response induced by M.cat. The results of these studies contribute to an increased understanding of molecular mechanism and possible novel treatment strategies for diseases caused by M.cat by specifically targeting TLRs and their signaling pathways.

Highlights

The innate immune system is critical for the initiation of effective immune response against invading pathogens
Lipopeptides and other components of Gram positive bacteria are recognized by TLR2 in conjunction with either TLR1 or TLR6, lipopolysacharride (LPS) is recognized by TLR4, flagellin is detected by TLR5, unmethylated DNA and CpG-oligodeoxynucleotides (CpG-DNA) by TLR9, double-stranded RNA or poly I:C by TLR3 and single stranded RNA by TLR7 and TLR8 [2]
BMMØ generated from CD142/2 and WT (C57BL/6J) mice were treated with M. cat LOS (100 ng/ml), E. coli LPS (100 ng/ml), the TLR2 ligand Pam3CSK4 (10 mg/ml), and live or HK M.cat for 6 hours and levels of TNF-a and IL-6 in culture supernatants were measured using enzyme-linked immunosorbent assay (ELISA)

Summary

Introduction

The innate immune system is critical for the initiation of effective immune response against invading pathogens. Toll like receptors (TLRs) are pattern recognition receptors that can ‘sense’ a wide range of microbial products, provoke host responses and produce various pro- and anti-inflammatory cytokines such as TNF-a, IL-6, IL-12, IFN-c, NO, IL-10 etc. Most of the receptors that detect bacterial products are located on cells surface (TLR2, TLR4, TLR5) while other receptors that can sense nucleic acid are located in intracellular compartments such as endosome (TLR3, TLR7, TLR8, TLR9) [3]. Once TLRs recognize microbial products, they activate distinct signaling pathways through different adaptor molecules such as MyD88, Mal, TRIF and TRAM, leading to the activation of various transcription factors (NF-kB, IRFs, AP-1 etc.) [4]. Among all the TLRs, only TLR4 has the unique ability to activate both MyD88-dependent and the TRIF-dependent signaling pathways, the later initiates the type-1 interferon response as well as late NF-kB activation [5], [6]

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Conclusion