Innate Immunity

Hyaluronan (HA) oligosaccharides stimulate pro-inflammatory responses in different cell types by modulating both cluster determinant 44 (CD44) and TLR4. The activation of these receptors is also mediated by collagen-induced arthritis (CIA) that, via two different pathways, culminates in the liberation of NF-κB. This then stimulates the production of pro-inflammatory cytokines, including IL-18 and IL-33, that are greatly involved in rheumatoid arthritis. The aim of this study was to investigate the effects of 6-mer HA oligosaccharides on mouse synovial fibroblasts obtained from normal DBA/J1 mice or mice subjected to CIA. Compared with normal synovial fibroblasts (NSF), rheumatoid arthritis synovial fibroblasts (RASF) showed no up-regulation of CD44 and TLR4 mRNA expression and the related proteins, as well as no activation of NF-κB. Very low levels of both mRNA and related proteins were also detected for IL-18 and IL-33. Treatment of NSF and RASF with 6-mer HA oligosaccharides significantly increased all the parameters in both fibroblast groups, although to a greater extent in RASF. The addition of hyaluronan binding protein to both NSF and RASF inhibited HA activity and was able to reduce the effects of 6-mer HA oligosaccharides and the consequent inflammatory response.

Epithelial to mesenchymal transition (EMT) has been linked to metastasis. Resveratrol exhibits potential antitumor activities; however, the inhibitory effects of resveratrol on the EMT of melanoma have not been demonstrated. Here, a new role for LPS in promoting EMT is described. LPS-induced EMT was identified by examining the markers of EMT. To assess the activation of NF-κB signal transduction pathway, we performed a reporter assay by using tumor cells transfected with the luciferase gene under the control of NF-κB response elements. The antitumor effects of resveratrol were evaluated in an experimental mouse metastasis tumor model. LPS increased N-cadherin and Snail expression and decreased zonula occludens-1 expression in a dose- and time-dependent manner. Meanwhile, LPS stimulated cell migration through activation of TLR4/NF-κB signal pathway. LPS-induced EMT is critical for inflammation-initiated metastasis. Nuclear translocation and transcriptional activity of p65 NF-κB, an important inducer of EMT, were inhibited by resveratrol. Resveratrol inhibited LPS-induced tumor migration and markers of EMT, significantly prolonged animal survival and reduced the tumor size. Thus, resveratrol plays an important role in the inhibition of LPS-induced EMT in mouse melanoma through the down-regulation of NF-κB activity. The data provide an insight into the mechanisms on the function of resveratrol during the processes of EMT.

Although the limulus amebocyte lysate (LAL) assay is widely used to determine the concentration of LPS in biological samples, it is known to be susceptible to interference caused by substances of non-bacterial origin. In particular, polysaccharides such as β-glucans and pectic polysaccharides from fungi or plants, respectively, were shown to give higher LPS readings than were actually present in the sample. Here, we describe an assay for the determination of LPS in biological samples based on the stimulation of TLR4/MD2/CD14 transfected HEK293 cells which dose dependently release IL-8 upon stimulation with increasing concentrations of highly purified Escherichia coli LPS. The resulting standard curve is used to determine the LPS concentration in unknown samples. We show that the outcome of the LPS stimulation is not affected by the presence of β-glucans or other environmental substances found in dust extracts. Although, we present evidence that the LPS concentration measured with the kinetic chromogenic LAL test correlates with data from the TLR4 assay, the LAL test displays higher LPS readings. We conclude that the described TLR4 assay is a reliable alternative to assess the concentration of LPS in environmental samples without being influenced by polysaccharides such as β-glucans and other environmental substances found in dust extracts.

We evaluated expression of TLR2, TLR4 and proinflammatory genes [NF-κB, TNF-α, cyclooxygenase-2 (COX-2)] in liver samples of patients in different stages of liver disease. Fifteen patients with unexplained transaminases elevation (reference group), 22 with viral chronic hepatitis (hepatitis group), 14 with virus-induced severe fibrosis/cirrhosis (cirrhosis group) and 10 with hepatocarcinoma (hepatocarcinoma group) were consecutively included in the study. Quantification of TLR2, TLR4, NF-κB, TNF-α and COX-2 mRNA was done by real-time RT-PCR and TLR2 and TLR4 protein expression was evaluated by immunohistochemistry. Compared with reference, TLR2 and TLR4 mRNA was increased in hepatitis (TLR2: 2.66 ± 0.69; TLR4: 3.11 ± 0.79; P < 0.05) and cirrhosis (TLR2: 2.14 ± 0.5; TLR4: 1.74 ± 0.27; P < 0.05) and decreased in hepatocarcinoma (TLR2: 0.48 ± 0.15; TLR4: 0.54 ± 0.10; P < 0.05). This associated with increased TNF-α and COX-2 mRNA in hepatitis (TNF-α: 3.24 ± 0.79; COX-2: 2.47 ± 0.36; P < 0.05) and cirrhosis (TNF-α: 1.73 ± 0.28; COX-2: 1.8 ± 0.35, P < 0.05), whereas NF-κB mRNA was increased in hepatitis (2.42 ± 0.31; P < 0.05) and unchanged in cirrhosis (1.34 ± 0.17; P = 0.3). Hepatocarcinoma presented increased COX-2 mRNA (1.63 ± 0.15; P < 0.05) and maintained (at decreased levels) mRNA of NF-κB (0.52 ± 0.12) and TNF-α (0.52 ± 0.12; P < 0.05, all genes). Immunohistochemistry confirmed increased expression of TLR2 and TLR4 in hepatitis and cirrhosis and maintained expression in hepatocarcinoma. Upregulation of TLR2, TLR4 and their proinflammatory mediators is associated with virus-induced hepatic IFC sequence.

Intense lung inflammation characterizes respiratory failure associated with Pneumocystis pneumonia. Our laboratory has previously demonstrated that alveolar epithelial cells (AECs) elaborate inflammatory cytokines and chemokines in response to the Pneumocystis carinii cell wall constituent β-(1→3)-glucan (PCBG), and that these responses require lactosylceramide, a prominent glycosphingolipid constituent of certain cell membrane microdomains. The relevance of membrane microdomains, also termed plasma membrane lipid rafts, in cell signaling and macromolecule handling has been increasingly recognized in many biologic systems, but their role in P. carinii-induced inflammation is unknown. To investigate the mechanisms of microdomain-dependent P. carinii-induced inflammation, we challenged primary rat AECs with PCBG with or without pre-incubation with inhibitors of microdomain function. Glycosphingolipid and cholesterol rich microdomain inhibition resulted in significant attenuation of P. carinii-induced expression of TNF-α and the rodent C-X-C chemokine MIP-2, as well as their known inflammatory secondary signaling pathways. We have previously shown that protein kinase C (PKC) is activated by PCBG challenge and herein show that PKC localizes to AEC microdomains. We also demonstrate by conventional microscopy, fluorescence microscopy, confocal microscopy and spectrophotofluorimetry that AECs internalize fluorescently-labeled PCBG by microdomain-mediated mechanisms, and that anti-microdomain pretreatments prevent internalization. Taken together, these data suggest an important role for AEC microdomain function in PCBG-induced inflammatory responses. This offers a potential novel target for therapeutics for a condition that continues to exert unacceptable morbidity and mortality among immunocompromised populations.

Organ failure is a severe complication in sepsis for which the pathophysiology remains incompletely understood. Recently, the matri-cellular cysteine-rich, angiogenic induced, 61 (Cyr61/CCN1); connective tissue growth factor (Ctgf/CCN2); and nephroblastoma overexpressed gene (Nov/CCN3) (CCN)-protein family have been attributed organ-protective properties. Their expression is sensitive to mediators of sepsis pathophysiology but a potential role in sepsis remains elusive. To provide an initial assessment, 50 rats were subjected to 18 h of cecal-ligation and puncture or sham operation. Hepatic and pulmonary CCN1 mRNA displayed an average 7.4- and 3.3-fold induction, while its cardiac expression was unchanged. The changes coincided with excessive hepatic and pulmonary inflammatory gene activation and a restricted cardiac inflammation. Furthermore, hepatocytes displayed a dosage-dependent CCN1 mRNA response in vitro, supporting a cytokine-mediated CCN1 regulation in sepsis. CCN2 mRNA was 2.2-fold induced in the liver, while 2.0-fold and 1.4-fold repressed in the heart and lung. Meanwhile, it did not respond to TNF-α exposure in vitro, which indicates different means of regulation than for CCN1. Taken together, this study provides the first evidence for multi-organ regulation of CCN1 and CCN2 in early stages of sepsis, and implies the eruption of inflammatory mediators as a potential mechanism behind the observed CCN1 regulation.

In innate immunity, dead and dying cells release internal constituents that can serve as damage-associated molecular patterns (DAMPs) or alarmins. This release occurs more abundantly during necrosis than apoptosis and may account for the differences in the immunologic properties of these death forms. To elucidate DAMP release in necrosis, we compared the levels of two nuclear molecules (DNA and HMGB1, a non-histone protein with alarmin activity) in media following necrosis of Jurkat T cells by freeze-thawing, ethanol, heat or hydrogen peroxide treatment. In our experiments, DNA release was measured by fluorimetry with the dye PicoGreen, while HMGB1 was measured by Western blotting. As the results of our study show, each form of necrosis is associated with a distinct pattern of DNA and HMGB1 release with respect to kinetics and amounts. Of these, freeze-thawing produced the highest and most rapid increase in HMGB1 and DNA levels, although the released DNA was subject to nuclease digestion; in addition, freeze-thawing led to the production of particles measured by flow cytometry. Together, these results indicate that experimental necrosis leads to diverse patterns of nuclear molecule release which could affect their immunologic activity.

Nicotinamide phosphoribosyltransferase (NAMPT), an enzyme involved in NAD biosynthesis, has recently been identified as a novel mediator of innate immunity.

In the present study, we report that treatment of LPS-primed monocytes with ATP greatly enhanced the secretion of NAMPT in a time- and concentration-dependent manner without displaying any cytotoxic effect. NAMPT release was suppressed by pretreatment with the P2X₇ receptor (P2X₇R) inhibitors oxidized ATP (oxATP) and KN-62, indicating the engagement of P2X₇Rs. Furthermore, P2X₇R was found to be involved in mediating cell permeability caused by the addition of ATP. To define a role of endogenous ATP in NAMPT secretion, LPS-primed monocytes were incubated in the presence of oxATP and KN-62, as well as the ATP-hydrolyzing enzymes apyrase and hexokinase. With the exception of oxATP, neither substance led to a decrease in NAMPT release, suggesting that autocrine/paracrine ATP is unlikely to be responsible for the LPS-induced release of NAMPT. In conclusion, the enhanced release of NAMPT by extracellular ATP described here indicates the requirement of a second stimulus for the efficient secretion of NAMPT. This mode of secretion, which also applies to IL-1β, might represent a general mechanism for the release of leaderless secretory proteins at locally restricted sites.

Innate immunity is crucial for an effective host defense against pathogenic microorganisms in periodontal tissues. As periodontal ligament (PDL) cells synthesize immunomodulatory cytokines, the aim of this in vitro study was to investigate whether these cells can interact with innate immune cells. Resting and inflammatory primed (IL-1β, TNF-α, HMGB1) human PDL cells were co-cultured with human monocyte-derived dendritic cells or macrophages. Migration, phenotypic maturation and modulation of phagocytosis of Porphyromonas gingivalis by immune cells were investigated upon co-culture with PDL cells and/or their released soluble factors. PDL cells interacted with immune cells under both non-inflammatory and inflammatory conditions. Immune cell migration was significantly enhanced by co-culture with PDL cells, which also affected their phenotypic maturation both through cell-cell contact and through released soluble mediators. The dendritic cell maturation markers CD83 and CD86 were upregulated as much as both ‘alternatively activated’ M2 macrophage maturation markers CD23 and CD163. In contrast, the ‘classically activated’ M1 macrophage maturation marker CD64 was downregulated. Finally, PDL cells significantly enhanced the phagocytosis of Porphyromonas gingivalis by immune cells. Our experiments revealed that PDL cells are not only structural elements of the periodontium, but actively influence immune responses by interaction with innate immune cells.

NF-κB, which comprises homo- and hetero-dimers of the five members of the Rel family, plays a crucial role in immunity to infection. The cRel and p50 subunits have been implicated in the development and function of the immune cells, but their in vivo importance remains poorly explored in sepsis. We aimed to study the impact of the combined loss of these two subunits on the innate response to infection in a cecal ligation and puncture model of sepsis. We have explored the possible defects in host defense, including pathogen clearance, bacterial phagocytosis and cytokine plasma release. We also performed gene profiling of cRel^−/−p50^−/− and wild-type LPS-stimulated peritoneal macrophages. Deficiency of cRel and p50 led to enhanced mortality to sepsis that was associated with defective macrophages phagocytosis, decreased bacterial clearance and moderate cytokine response. Transcription profile analysis revealed a common inflammatory response but a significant down-regulated transcription of genes encoding for pathogen recognition receptors and antimicrobial molecules, supporting the in vivo findings in mice. In conclusion, the cRel and p50 subunits of NF-κB play an important combined role in the innate response and are crucial for survival and pathogen clearance in polymicrobial sepsis.

Modification of a lipid A moiety in Gram-negative bacterial LPS to a less acylated form is thought to facilitate bacterial evasion of host innate immunity, thereby enhancing pathogenicity. The contribution of less-acylated lipid A to interactions of whole bacterial cells with host cells (especially in humans) remains unclear. Mutant strains of Salmonella enterica serovar Typhimurium with fewer acylated groups were generated. The major lipid A form in wild-type (WT) and the mutant KCS237 strain is hexa-acylated; in mutant strains KCS311 and KCS324 it is penta-acylated; and in KCS369 it is tetra-acylated. WT and KCS237 formalin-killed and live bacteria, as well as their LPS, strongly stimulated production of pro-inflammatory cytokines in human U937 cells; this stimulation was suppressed by TLR4 suppressors. LPS of other mutants produced no agonistic activity, but strong antagonistic activity, while their formalin-killed and live bacteria preparations had weak agonistic and no antagonistic activity. Moreover, these less-acylated mutants had increased resistance to phagocytosis by U937 cells. Our results indicate that a decrease of one acyl group (from six to five) is enough to allow Salmonella to evade human innate immunity and that the antagonistic activity of less-acylated lipid A is not utilized for this evasion.

Koch’s postulates have shaped our understanding of infectious diseases; however, one of the tangential consequences of them has been the emergence of a predominantly monomicrobial perspective concerning disease aetiology. This orthodoxy has been undermined by the growing recognition that some important infectious diseases have a polymicrobial aetiology. A significant new development in our understanding of polymicrobial infections is the recognition that they represent functional ecosystems and that to understand such systems and the outcome and impact of therapeutic interventions requires an understanding of how these communities arise and develop. Therefore, it is timely to explore what we can learn from other fields. In particular, ecological theory may inform our understanding of how polymicrobial communities assemble their structure and their dynamics over time. Such work may also offer insights into how such communities move from stable to unstable states, as well as the role of invasive pathogens in the progression of the disease. Ecological theory offers a theoretical framework around which testable hypotheses can be developed to clarify the polymicrobial nature and dynamics of such infections in the face of environmental change and therapeutic interventions.