Innate Immunity

The molecular mechanisms that have been designed to protect the host from invading pathogens are responsible for sepsis, an often fatal response of the immune system against microbial pathogens. In the past few years, intense research in the field of innate immunity has identified a plethora of pattern recognition receptors that are responsible for bacterial-induced activation. Recognition of bacterial lipopolysaccharide seems to involve a complex orchestration of protein—protein interactions that eventually leads to cellular activation. In this review, we attempt to unravel the dynamic interactions that occur among the different receptors involved and dictate the outcome of the innate immune response.

The fimA gene encodes the major fimbrial protein of Porphyromonas gingivalis. It has been shown to stimulate adhesion to salivary proteins and other bacteria. It is also thought to play a major role in invading and stimulating host cells. To determine whether the fimA gene represents one of the principal molecules of P. gingivalis that induces inflammation, we tested purified FimA protein and a mutant P. gingivalis (DPG3) that lacks the fimA gene versus wild-type (WT) P. gingivalis. When injected into connective tissue of the scalp, purified FimA protein induced TNF-α and MIP-2 expression confirming that it is pro-inflammatory. WT P. gingivalis induced TNF-α expression and recruitment of PMNs in the same model. However, DPG3 P. gingivalis stimulated TNF expression and PMN recruitment to the same extent. The latter was consistent with similar induction of the chemokine MIP-2. Similar results were obtained with diabetic mice that have a more prolonged inflammatory response to bacterial stimulation. These results indicate that FimA is a potent inducer of inflammatory cytokine expression but, in the context of P. gingivalis infection, it is not a principal stimulator of the innate host response.

Background: We have previously shown that chylomicron (CM)-bound lipopolysaccharide (LPS) inhibits the host innate immune response by rendering hepatocytes tolerant to pro-inflammatory cytokine stimulation. However, LPS is a complex macromolecule containing both lipid and carbohydrate domains. We hypothesized that just as lipid A confers the toxicity of LPS, it is also responsible for the immunoregulatory effect on hepatocytes.

Methods: We pretreated primary rat hepatocytes for 2 h with a series of CM-LPS complexes in which the endotoxin moiety varied in its structure and/or toxicity. Subsequently, the cells were stimulated with a mixture of pro-inflammatory cytokines. Nitric oxide production was measured as an indicator of hepatocellular activation.

Results: All pretreatments wherein the CM-bound complex contained the lipid A moiety readily inhibited the hepatocellular cytokine response, including CM bound to lipid A alone. In contrast, CM-LPS complexes containing detoxified LPS, which lacks the lipid A domain, had no effect on the hepatocellular response to cytokines.

Conclusions: The lipid A domain of the LPS macromolecule is both sufficient and essential for the CM-mediated induction of cytokine tolerance in hepatocytes. However, this process is independent of the specific endotoxic activity of the lipid A moiety.

The rabbit test to detect pyrogenic contamination in parenterals is crucial to ensure patient safety. The pharmacopoeial tests in Europe, the US and Japan are based on the fever reaction of rabbits, but differ in their experimental design and in their algorithms to assess contamination. Employing an international reference endotoxin, fever can be induced in rabbits. Data from 171 rabbits built the base for probabilistic modelling of the fever reaction and for the comparison of the pharmacopoeial tests. The rabbit fever reaction could be modelled as a function of the amount of injected endotoxin (per kg body weight) by linear regression. Combining the pharmacopoeial algorithms of the rabbit pyrogen test with the developed model allowed analysis of differences regarding test results and animal consumption. This showed that the assessment of pyrogenic contamination strongly depends on the respective pyrogen test stipulated by regulations. Additionally, the approach was used to develop a new experimental design. Two specific versions of this design resulted in a reduction of the number of animals used by about 30% while the safety of the test was maintained. A need for harmonisation is evident, allowing optimisation of the experimental design, which promotes animal welfare.

Postoperative infections after hepatectomy sometimes lead to fatal hepatic failure, but the mechanism of the hepatic failure is unclear. Wistar rats underwent 90% hepatectomy, and were then divided into three groups: (i) the SAL group, injected with normal saline; (ii) the LPS group, injected with lipopolysaccharide (LPS) every day for 1 week; and (iii) the LPS plus TGF-Ab (LPS+TGF-Ab) group, injected with LPS with anti-transforming growth factor-β1 (TGF-β1) antibody. We investigated survival rates, TGF-β1 expression in the liver, liver regeneration by proliferating cell nuclear antigen labeling index, hepatocyte apoptosis by single stranded DNA labeling index, and perisinusoidal fibrosis using Masson's trichrome staining. The LPS group (30.4%) had a significantly lower survival rate than the SAL group (84%) and tended to be lower than the LPS+TGF-Ab group (49.4%). Liver regeneration in the LPS group was significantly lower than in the other groups. In the LPS group, hepatocyte apoptosis and perisinusoidal fibrosis was significantly more remarkable, and TGF-β1 expression was significantly higher than in the SAL group. TGF-β1 enhanced by LPS plays an important role in the mechanism of hepatic failure by infections after hepatectomy, especially in inhibition of liver regeneration, and induction of hepatocyte apoptosis and perisinusoidal fibrosis.

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind, and in some cases hydrolyse, bacterial peptidoglycans (PGNs). We determined the crystal structure of the C-terminal PGN-binding domain of human PGRP-Iα in complex with a muramyl tripeptide representing the conserved core of lysine-type PGNs. The peptide stem of the ligand is buried at the deep end of a long binding groove, with N-acetylmuramic acid situated in the middle of the groove, whose shallow end could accommodate N-acetylglucosamine. Both peptide and glycan moieties are essential for binding by PGRPs. Conservation of key PGN-contacting residues indicates that all PGRPs employ this basic PGN-binding mode. The structure identifies variable residues that likely mediate discrimination between lysine- and diaminopimelic acid-type PGNs. In addition, we propose a mechanism for PGN hydrolysis by Zn²⁺-containing catalytic PGRPs.

Small mannose-binding lectin (MBL)-associated protein (sMAP) is a component of the complex consisting of MBL and MBL-associated serine proteases (MASPs) in the lectin complement pathway. sMAP is a truncated form of MASP-2, which is generated by an alternative splicing from a single structural MASP-2 gene. Upon activation of the MBL—MASPs complex, MASP-2 cleaves the complement C4, but the role of sMAP which lacks the serine protease domain is not clear. To clarify the role of sMAP in activation of the lectin pathway, we have generated sMAP-gene deficient mice which are also deficient for MASP-2. In this study, we generated and purified mouse recombinant sMAP (rsMAP) and rMASP-2 using the Drosophila expression system for the reconstitution assay of the deficient mice. In preliminary experiments, these purified recombinants were able to reconstitute the MBL—MASPs—sMAP complexes and the addition of rMASP-2 to deficient serum restored the C4 cleavage activity of the MBL—MASPs complex. From these data, rsMAP and rMASP-2 generated in this study seem to be useful in analysis of the deficient mice.

Toll-like receptors (TLRs) recognize specific components of micro-organisms and trigger the activation of innate immunity. TLR-mediated signaling pathways are now rapidly being elucidated. TLR signaling originates from the cytoplasmic Toll/IL-1 receptor (TIR) domain, which is conserved among all TLRs. Furthermore, TIR domain-containing adaptors, such as MyD88, TRIF, TIRAP and TRAM, play essential roles in TLR signaling. MyD88 is essential for inflammatory cytokine production via all TLRs, whereas TRIF is involved in TLR3- and TLR4-mediated MyD88-independent induction of IFN-β. Thus, innate immunity represents a skilful system that senses microbial invasion and initiates appropriate immune responses.

The Salmonella enterica serovar Typhimurium virulence gene products PhoP/PhoQ sense host microenvironments to regulate the expression of a lipid A 3-O-deacylase, PagL, and a lipid A palmitoyltransferase, PagP. Therefore, deacylation and/or palmitoylation of lipid A could occur in Salmonellae adapted to host environments. The PhoP/PhoQ-regulated modification of lipid A alters host recognition and signaling, and may play an important role in host defense against Salmonellae infection. Here we report the purification and characterization of modified lipid A species. Deacylated lipid A, deacylated and palmitoylated lipid A, and palmitoylated lipid A species were generated in Escherichia coli cells heterologously expressing salmonellae PagL and/or PagP, and then purified by sequential thin-layer chromatography. The purified lipid A species showed m/z values that correspond to single lipid A species on mass spectrometry analysis. The modified lipid A species showed reduced ability to induce cellular signaling through Toll-like receptor 4, suggesting a specific function of the lipid A modifications in the pathogenesis of salmonellae infection.