Innate Immunity

Impaired resistance to Pseudomonas aeruginosa-induced pneumonia after cecal ligation and puncture (CLP), a mouse model for human polymicrobial sepsis, is associated with decreased IFN-γ, but increased IL-10, levels in the lung. We investigated the so far unknown mechanisms underlying this reduced IFN-γ synthesis in CLP mice. CD11b⁺ NK cells, but not T or NKT cells in the lung were impaired in IFN-γ synthesis upon challenge with Pseudomonas in vitro and in vivo after CLP. The inhibition of NK cells was independent of IL-10. IFN-γ synthesis of NK cells was only partly restored by addition of recombinant IL-12. Accessory cells including dendritic cells and alveolar macrophages were required for maximal IFN-γ secretion. But accessory cells of CLP mice suppressed the IFN-γ secretion from naive lung leukocytes. In turn, naive accessory cells were unable to restore the IFN-γ production from lung leukocytes of CLP mice. Thus, a disturbed interaction of accessory cells and NK cells is involved in the impaired IFN-γ release in response to Pseudomonas in the lung of CLP mice. Considering the importance of IFN-γ in the immune defense against bacteria the dysfunction of accessory cells and NK cells might contribute to the enhanced susceptibility to Pseudomonas after CLP.

During Helicobacter pylori (Hp) infections, innate immune cells may be positively or negatively modulated by Hp compounds or by Hp-induced cytokines. We have shown previously that the natural cytotoxic activity of PBMC was lower in Hp-infected [Hp(+)] than Hp-uninfected individuals [Hp(−)]. Here, we asked whether the Hp-modulated cytotoxic amplitude is associated with changes in the number of NK cells, their activation or intracellular cytokine expression. Flow cytometry immunophenotyping of PBMC was performed with regard to the surface receptors CD3, CD56 and CD25, and intracellular cytokine expression of IL-2, IFN-γ and IL-10 after in vitro stimulation with Hp glycine acid extract (GE), Hp LPS or standard Escherichia coli LPS. Hp GE-driven enhancement of lymphocyte cytotoxic activity was associated with the expansion of CD3⁻CD56⁺CD25⁺ NK cells and the up-regulation of IFN-γ and/or IL-2 synthesis, up to the higher level in Hp(−) than in Hp(+), while Hp LPS-mediated decrease in lymphocyte cytotoxicity was accompanied by the lack of CD3⁻CD56⁺CD25⁺ NK propagation, the inhibition of pro-inflammatory cytokine expression and intense expansion of IL-10–producing NK cells. Thus, the cytotoxic and cytokine activities of NK cells were dependent on the type of antigenic challenge and the Hp status, that is, NK cells could be modulated positively by Hp GE Ags and negatively by Hp LPS.

Burn induces an immunopathological response involving multiple immune cell types that includes γδ T-cells. Nonetheless, the role of γδ T-cells at the wound site after burn is not clearly defined. Wild type and γδ T-cell receptor deficient (δ TCR^-/-) mice were subjected to a major burn or sham procedure. At 1–7 d thereafter, skin samples were collected and T-cell populations analyzed. The majority of T-cells in the skin of sham mice were γδ T-cells. After burn, however, an increase in the total T-cells was observed at the wound site and these cells were predominantly αβ T-cells. Their influx was γδ T-cell dependent, as it was markedly reduced in injured δ TCR^-/- mice. Burn wound γδ T-cells were activated with increased expression of TLRs and CD69. In contrast, the infiltrating αβ T-cells TLR and CD69 expressions were attenuated after burn. Thus, burn is associated with of γδ T-cell activation at the injury site, which initiates a massive infiltration of the wound with αβ T-cells that likely facilitate the transition from the inflammatory to the proliferative phase of healing.

Asymptomatic carriage of Campylobacter jejuni is highly prevalent in chicken flocks. Thus, we investigated whether chronic Campylobacter carriage affects chicken intestinal functions despite the absence of clinical symptoms. An experiment was carried out in which commercial chickens were orally infected with C. jejuni (1 × 10⁸ CFU/bird) at 14 days of life. Changes in ion transport and barrier function were assessed by short-circuit current (I_sc) and transepithelial ion conductance (G_t) in Ussing chambers. G_t increased in cecum and colon of Campylobacter-infected chicken 7 d post-infection (DPI), whereas G_t initially decreased in the jejunum at 7 DPI and increased thereafter at 14 DPI. The net charge transfer across the epithelium was reduced or tended to be reduced in all segments, as evidenced by a decreased I_sc. Furthermore, the infection induced intestinal histomorphological changes, most prominently including a decrease in villus height, crypt depth and villus surface area in the jejunum at 7 DPI. Furthermore, body mass gain was decreased by Campylobacter carriage. This study demonstrates, for the first time, changes in the intestinal barrier function in Campylobacter-infected chickens and these changes were associated with a decrease in growth performance in otherwise healthy-appearing birds.

TLRs represent one of the most important components of innate immunity. Currently, these receptors have been extensively studied in vertebrates and insects, but our knowledge for annelids is very limited. Therefore, the aim of our study was to identify earthworm TLR homologs by genome-wide screening, and to determine the expression of candidate genes as a response to Gram-positive and Gram-negative bacteria. Using a combination of deep pyrosequencing and quantitative PCR we found six candidate genes, for which all were expressed in Dentrobaena veneta. Two of the candidates showed significant response to bacterial exposure. In conclusion, TLRs seem to have a role in earthworm immunology.

The aims of this study were to investigate OX40 ligand expression in sinus tissue from patients with nasal polyposis compared with patients with chronic rhinosinusitis without nasal polyps (NPs), and to determine if OX40 ligand expression is related to eosinophilic sinus infiltration. Twenty patients with chronic rhinosinusitis (11 with and nine without NPs) and seven controls were enrolled in the study. The mRNA expression of OX40 ligand and thymic stromal lymphopoietin and its receptor were analyzed. The immunoreactivity score for OX40 ligand and the eosinophil count were obtained. The mRNA expression and immunoreactivity score of OX40 ligand were higher in patients with nasal polyposis than in patients without NPs, as well as healthy controls. The mRNA expression of thymic stromal lymphopoietin and its receptor was significantly higher in nasal polyposis than in the control, but not significantly higher than in chronic rhinosinusitis without NPs. A correlation between the number of OX40 ligand-positive cells and the number of eosinophils in sinus biopsies was found only in patients with nasal polyposis. In conclusion, the thymic stromal lymphopoietin/OX40 ligand axis is up-regulated in nasal polyposis and is related to the intensity of eosinophilic inflammation.

TLRs are key innate immune receptors that recognize conserved features of biological molecules that are found in microbes. In particular, TLR2 has been reported to be activated by different kinds of microbial ligands. To advance our understanding of the interaction of TLR2 with its ligands, the recombinant human TLR2 ectodomain (hTLR2ED) was expressed using a baculovirus/insect cell expression system and its biochemical, as well as ligand binding, properties were investigated. The hTLR2ED binds synthetic bacterial and mycoplasmal lipopeptides, lipoteichoic acid from Staphylococcus aureus, and synthetic lipoarabinomannan precursors from Mycobacterium at extracellular physiological conditions, in the absence of its co-receptors TLR1 and TLR6. We also determined that lipopeptides and glycolipids cannot bind simultaneously to hTLR2ED and that the phosphatidyl inositol mannoside 2 (Pim2) is the minimal lipoarabinomannan structure for binding to hTLR2ED. Binding of hTLR2ED to Pim4, which contains a diacylglycerol group with one of its acyl chains containing 19 carbon atoms, indicates that hTLR2ED can bind ligands with acyl chains longer than 16 carbon atoms. In summary, our data indicate that diacylglycerol is the ligand moiety of microbial glycolipids and lipoproteins that bind to hTLR2ED and that both types of ligands bind to the same binding site of hTLR2ED.

Here we report that LPS induces osteoclast (OC) formation in murine RAW 264.7 macrophage cells in RPMI-1640 medium but not in α-minimum essential medium (α-MEM) as the original culture medium. LPS-induced OC formation in both media was examined to clarify the differential response. Receptor activator of NF-κB ligand induced OC formation in either α-MEM or RPMI-1640 medium. However, LPS-induced OC formation in RAW 264.7 cells maintained in RPMI-1640 medium, but not α-MEM, which was also supported by mouse bone marrow-derived macrophages, although they were less sensitive to LPS than RAW 264.7 cells. LPS augmented the expression of nuclear factor of activated T-cells (NFATc1) as a key transcription factor of osteoclastogenesis in cells maintained in RPMI-1640 medium, but reduced it in cells maintained in α-MEM. A high concentration of LPS was cytotoxic against cells maintained in α-MEM. Glutathione exclusively present in RPMI-1640 medium prevented LPS-induced cell death in α-MEM and augmented LPS-induced NFATc1 expression, followed by enhanced LPS-induced OC formation. LPS induced higher generation of reactive oxygen species in α-MEM than RPMI-1640 medium. An antioxidant enhanced LPS-induced OC formation, whereas a pro-oxidant reduced it. Taken together, redox balance in the culture condition was suggested to regulate in vitro LPS-induced OC formation.

This study was designed to understand the contribution of the inflammasome and IL-1β activation in otitis media (OM). We examined the middle ear (ME) response to non-typeable Haemophilus influenzae (NTHi) in wild type (WT) mice using gene microarrays and a murine model of acute OM. Expression of members of the NOD domain-like receptor family of inflammasome genes was significantly up-regulated early in NTHi infection of the ME, potentially activating specific downstream regulatory cascades that contribute to the proliferative inflammatory response observed during OM. Expression of the pro-forms of the inflammasome targets IL-1β and IL-18 were also up-regulated. To evaluate the role of inflammasome-mediated cytokine maturation, NTHi-induced OM was examined in Asc^−/−-deficient mice and compared with that seen in WT mice. Mice lacking the Asc gene showed near absence of IL-1β maturation in the ME and a reduction in leukocyte recruitment and infiltration to the cavity, and their macrophages exhibited reduced phagocytosis of NTHi. These inflammatory defects were linked to an increase in the degree and duration of mucosal epithelial hyperplasia in the ME of Asc^−/− mice, as well as a delay in bacterial clearance from their MEs. These data demonstrate an important role for the inflammasome and cytokine processing in the course and resolution of OM.

Otitis media (OM), one of the most prevalent diseases in young children, is clinically important owing to its high incidence in children and its potential impact on language development and motor coordination. OM is the most common reason for the prescription of antibiotics (accounting for 25% of prescriptions) due to its extremely high incidence. A recent increase in antibiotic resistance among OM pathogens is emerging as a major public health concern globally, which led us to consider non-antibiotic approaches for the management of OM. In this study, we evaluated gene transfer of an antimicrobial peptide, human β-defensin 2 (DEFB4), using an adenoviral vector (Ad5 with deletions of E1/E3/E4) as a potential therapeutic approach. We demonstrated that the transduction of human β-defensin 2 induces the production of human β-defensin 2 and suppresses non-typeable Haemophilus influenzae (NTHi) adhesion to human middle ear epithelial cells. Moreover, intratympanic inoculation of Ad-DEFB4 was found to attenuate NTHi-induced middle ear effusions without eliciting a significant immune response. Most importantly, intratympanic inoculation of Ad-DEFB4 appeared to significantly augment clearance of NTHi from middle ear cavity. Collectively, our results suggest that intratympanic gene delivery of antimicrobial molecules may serve as an alternative/adjuvant approach for the management of OM.