Involvement of endoplasmic reticulum and histone proteins in immunomodulation by TLR4-interacting SPA4 peptide against Escherichia coli.

Pulmonary host defense is critical for the control of lung infection and inflammation. An increased expression and activity of Toll-like receptor 4 (TLR4) induce phagocytic uptake/clearance and inflammation against Gram-negative bacteria. In this study, we addressed the mechanistic aspect of the immunomodulatory activity of the TLR4-interacting SPA4 peptide (amino acid sequence GDFRYSDGTPVNYTNWYRGE) against Escherichia coli. Binding of the SPA4 peptide to bacteria and direct anti-bacterial effects were investigated using flow cytometric, microscopic, and bacteriological methods. The bacterial uptake and inflammatory cytokine response were studied in dendritic cells expressing endogenous basal level of TLR4 or overexpressing TLR4. The subcellular distribution and co-localization of TLR4 and bacteria were investigated by immunocytochemistry. Furthermore, we studied the cellular expression and co-localization of endoplasmic reticulum (ER) molecules (calnexin and ER membrane protein complex subunit 1; EMC1) with lysosomal-associated membrane protein 1 (LAMP1) in cells infected with E. coli and treated with the SPA4 peptide. Simultaneously, the expression of histone H2A protein was quantitated by immunoblotting. Our results demonstrate no binding or direct killing of the bacteria by SPA4 peptide. Instead, it induces the uptake and localization of E. coli in the phagolysosomes for lysis and simultaneously suppresses the secreted levels of TNF-α. Overexpression of TLR4 further augments the pro-phagocytic and anti-inflammatory activity of SPA4 peptide. A time-dependent change in subcellular distribution of TLR4 and an increased co-localization of TLR4 with E. coli in SPA4 peptide-treated cells suggest an enhanced recognition and internalization of bacteria in conjugation with TLR4. Furthermore, an increased co-localization of calnexin and EMC1 with LAMP1 indicates the involvement of ER in pro-phagocytic activity of SPA4 peptide. Simultaneous reduction in secreted amounts of TNF-α coincides with suppressed histone H2A protein expression in the SPA4 peptide-treated cells. These results provide initial insights into the plausible role of ER and histones in the TLR4-immunomodulatory activity of SPA4 peptide against Gram-negative bacteria.

Lung and general health effects of Toll-like receptor-4 (TLR4)-interacting SPA4 peptide.

BACKGROUND: A surfactant protein-A-derived peptide, which we call SPA4 peptide (amino acids: GDFRYSDGTPVNYTNWYRGE), alleviates lung infection and inflammation. This study investigated the effects of intratracheally administered SPA4 peptide on systemic, lung, and health parameters in an outbred mouse strain, and in an intratracheal lipopolysaccharide (LPS) challenge model. METHODS: The outbred CD-1 mice were intratracheally administered with incremental doses of SPA4 peptide (0.625-10 µg/g body weight) once every 24 h, for 3 days. Mice left untreated and those treated with vehicle were included as controls. Mice were euthanized after 24 h of last administration of SPA4 peptide. In order to assess the biological activity of SPA4 peptide, C57BL6 mice were intratracheally challenged with 5 µg LPS/g body weight and treated with 50 µg SPA4 peptide via intratracheal route 1 h post LPS-challenge. Mice were euthanized after 4 h of LPS challenge. Signs of sickness and body weights were regularly monitored. At the time of necropsy, blood and major organs were harvested. Blood gas and electrolytes, serum biochemical profiles and SPA4 peptide-specific immunoglobulin G (IgG) antibody levels, and common lung injury markers (levels of total protein, albumin, and lactate, lactate dehydrogenase activity, and lung wet/dry weight ratios) were determined. Lung, liver, spleen, kidney, heart, and intestine were examined histologically. Differences in measured parameters were analyzed among study groups by analysis of variance test. RESULTS: The results demonstrated no signs of sickness or changes in body weight over 3 days of treatment with various doses of SPA4 peptide. It did not induce any major toxicity or IgG antibody response to SPA4 peptide. The SPA4 peptide treatment also did not affect blood gas, electrolytes, or serum biochemistry. There was no evidence of injury to the tissues and organs. However, the SPA4 peptide suppressed the LPS-induced lung inflammation. CONCLUSIONS: These findings provide an initial toxicity profile of SPA4 peptide. Intratracheal administration of escalating doses of SPA4 peptide does not induce any significant toxicity at tissue and organ levels. However, treatment with a dose of 50 µg SPA4 peptide, comparable to 2.5 µg/g body weight, alleviates LPS-induced lung inflammation.

Effect of liposome-encapsulated hemoglobin resuscitation on proteostasis in small intestinal epithelium after hemorrhagic shock.

Gut barrier dysfunction is the major trigger for multiorgan failure associated with hemorrhagic shock (HS). Although the molecular mediators responsible for this dysfunction are unclear, oxidative stress-induced disruption of proteostasis contributes to the gut pathology in HS. The objective of this study was to investigate whether resuscitation with nanoparticulate liposome-encapsulated hemoglobin (LEH) is able to restore the gut proteostatic mechanisms. Sprague-Dawley rats were recruited in four groups: control, HS, HS+LEH, and HS+saline. HS was induced by withdrawing 45% blood, and isovolemic LEH or saline was administered after 15 min of shock. The rats were euthanized at 6 h to collect plasma and ileum for measurement of the markers of oxidative stress, unfolded protein response (UPR), proteasome function, and autophagy. HS significantly increased the protein and lipid oxidation, trypsin-like proteasome activity, and plasma levels of IFNγ. These effects were prevented by LEH resuscitation. However, saline was not able to reduce protein oxidation and plasma IFNγ in hemorrhaged rats. Saline resuscitation also suppressed the markers of UPR and autophagy below the basal levels; the HS or LEH groups showed no effect on the UPR and autophagy. Histological analysis showed that LEH resuscitation significantly increased the villus height and thickness of the submucosal and muscularis layers compared with the HS and saline groups. Overall, the results showed that LEH resuscitation was effective in normalizing the indicators of proteostasis stress in ileal tissue. On the other hand, saline-resuscitated animals showed a decoupling of oxidative stress and cellular protective mechanisms.

Hemorrhage-induced interleukin-1 receptor pathway in lung is suppressed by 3,5-bis(2-fluorobenzylidene)-4-piperidone in a rat model of hypovolemic shock.

Severe blood loss in victims of trauma creates an exaggerated inflammatory background that contributes to the development of intravascular coagulopathy and multiple organ dysfunction syndrome. We hypothesized that treatment with diphenyldifluoroketone EF24, an inhibitor of nuclear factor kappa-B, would have salutary effects in hemorrhagic shock. The objective of this study was to investigate the effect of EF24 on the expression of the interleukin-1 receptor (IL-1R) superfamily in a rat model of hypovolemic shock. Hypovolemia was induced by gradually withdrawing approximately 50% of circulating blood, and EF24 was administered intraperitoneally (0.2 mg/kg) in 50 µL of saline. After 6 h of shock, lung tissue was probed immunohistochemically and by immunoblotting to study the expression of Toll-like receptor 4 (TLR4), IL-1R, suppression of tumorigenicity 2 (ST2), and single immunoglobulin IL-1R-related (SIGIRR). The tissue-associated pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α) and IL-6, were measured by enzyme-linked immunosorbent assay. We observed a reduction in immunoreactive TLR4 and IL-1R1 in lung tissue of rats treated with EF24. Simultaneously, the pulmonary expression of ST2 and SIGIRR (the putative down-regulators of the pro-inflammatory IL-1R pathway) was increased in EF24-treated hemorrhaged rats. The concentration of hemorrhage-induced TNF-α and IL-6 in lung tissue homogenates was also reduced by EF24 treatment. These results confirm our previous in vitro observations in lipopolysaccharide-stimulated dendritic cells that EF24 beneficially modulates the IL-1R pathway and suggest that it could be investigated as an adjunct therapeutic in managing inflammation associated with hemorrhagic shock.

EF24 suppresses maturation and inflammatory response in dendritic cells.

Synthetic curcuminoid EF24 was studied for its effect on the maturation and inflammatory response in murine bone marrow derived immortalized JAWS II dendritic cells (DCs). EF24 reduced the expression of LPS-induced MHC class II, CD80 and CD86 molecules. It also abrogated the appearance of dendrites, a typical characteristic of mature DCs. These effects were accompanied by the inhibition of LPS-induced activation of transcription factor nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB). Simultaneous reduction of pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, IL-6] both at the mRNA and secreted levels was also observed. To investigate the dependency of LPS effects on MyD88 adaptor protein, we transfected JAWS II DCs with dominant negative MyD88 plasmid construct (MyD88-DN). EF24 reduced NF-κB activity and TNF-α secretion in a MyD88-dependent manner. These results suggest that EF24 modulates DCs by suppressing their maturation and reducing the secretion of inflammatory cytokines. Further, it appears that EF24 acts at or upstream of MyD88 in the LPS-TLR4/MyD88/NF-κB pathway.

Stability and structure-activity relationship of the SPA4 peptide under ambient and stressed conditions of lung injury.

Lung inflammation and injuries are major health problems. The SPA4 peptide (amino acid sequence GDFRYSDGTPVNYTNWYRGE) binds to Toll-like receptor-4 and exerts anti-inflammatory activity. In this study, we have determined the stability of the structure and structure-activity relationship of the SPA4 peptide under ambient and stressed conditions of lung injury. The SPA4 peptide was maintained at different pH and temperatures, in solutions of different ionic strengths, and simulated lung fluids. The primary and secondary structure of the SPA4 peptide was determined by ultraviolet-visible (UV-VIS) and circular dichroism (CD) spectroscopy. The activity of the SPA4 peptide was determined by measurement of secreted levels of chemokine C-X-C motif ligand 1/keratinocyte-derived chemokine (CXCL1/KC) and lactate by primary mouse lung epithelial cells against lipopolysaccharide (LPS) stimuli. Our results demonstrate the stability of the structure of the SPA4 peptide at room temperature and 4 °C over 10 days. The original UV-VIS spectra of the SPA4 peptide followed a typical pattern when incubated in solutions of pH 5.7, 7.0, and 8.0 at different temperatures, simulated lung fluids, and most of the chemical components. Slight shifts in the absorbance peaks, derivative values, and vibrational fine structures were noted in the fourth-derivative spectra of the SPA4 peptide under some conditions. An increased level of lactate is the hallmark of lung injury. The SPA4 peptide on its own and in the presence of lactate exerts anti-inflammatory activity. The primary and secondary structure and the activity of the SPA4 peptide remain intact when pre-incubated in 2 mM sodium lactate solution. The results provide important insights about the stability and structure-activity relationship of the SPA4 peptide.

Distinct compartmentalization of NF-κB activity in crypt and crypt-denuded lamina propria precedes and accompanies hyperplasia and/or colitis following bacterial infection.

Citrobacter rodentium induces transmissible murine colonic hyperplasia (TMCH) and variable degrees of inflammation and necrosis depending upon the genetic background. Utilizing C. rodentium-induced TMCH in C3H/HeNHsd inbred mice, we observed significant crypt hyperplasia on days 3 and 7 preceding active colitis. NF-κB activity in the crypt-denuded lamina propria (CLP) increased within 24 h postinfection, followed by its activation in the crypts at day 3, which peaked by day 7. Increases in interleukin-α1 (IL-1α), IL-12(p40), and macrophage inflammatory protein 1α (MIP-1α) paralleled NF-κB activation, while increases in IL-1α/ß, IL-6/IL-12(p40)/granulocyte colony-stimulating factor (G-CSF)/keratinocyte-derived chemokine (KC)/monocyte chemotactic protein 1 (MCP-1), and MIP-1α followed NF-κB activation leading to significant recruitment of neutrophils to the colonic mucosa and increased colonic myeloperoxidase (MPO) activity. Phosphorylation of the crypt cellular and nuclear p65 subunit at serines 276 and 536 led to functional NF-κB activation that facilitated expression of its downstream target, CXCL-1/KC, during TMCH. Distinct compartmentalization of phosphorylated extracellular signal-regulated kinase 1 and 2 ([ERK1/2] Thr(180)/Tyr(182)) and p38 (Thr(202)/Tyr(204)) in the CLP preceded increases in the crypts. Inhibition of ERK1/2 and p38 suppressed NF-κB activity in both crypts and the CLP. Dietary administration of 6% pectin or 4% curcumin in C. rodentium-infected mice also inhibited NF-κB activity and blocked CD3, F4/80, IL-1α/ß, G-CSF/MCP-1/KC, and MPO activity in the CLP while not affecting NF-κB activity in the crypts. Thus, distinct compartmentalization of NF-κB activity in the crypts and the CLP regulates crypt hyperplasia and/or colitis, and dietary intervention may be a novel strategy to modulate NF-κB-dependent protective immunity to facilitate crypt regeneration following C. rodentium-induced pathogenesis.

Bacillus anthracis lethal toxin reduces human alveolar epithelial barrier function.

The lung is the site of entry for Bacillus anthracis in inhalation anthrax, the deadliest form of the disease. Bacillus anthracis produces virulence toxins required for disease. Alveolar macrophages were considered the primary target of the Bacillus anthracis virulence factor lethal toxin because lethal toxin inhibits mouse macrophages through cleavage of MEK signaling pathway components, but we have reported that human alveolar macrophages are not a target of lethal toxin. Our current results suggest that, unlike human alveolar macrophages, the cells lining the respiratory units of the lung, alveolar epithelial cells, are a target of lethal toxin in humans. Alveolar epithelial cells expressed lethal toxin receptor protein, bound the protective antigen component of lethal toxin, and were subject to lethal-toxin-induced cleavage of multiple MEKs. These findings suggest that human alveolar epithelial cells are a target of Bacillus anthracis lethal toxin. Further, no reduction in alveolar epithelial cell viability was observed, but lethal toxin caused actin rearrangement and impaired desmosome formation, consistent with impaired barrier function as well as reduced surfactant production. Therefore, by compromising epithelial barrier function, lethal toxin may play a role in the pathogenesis of inhalation anthrax by facilitating the dissemination of Bacillus anthracis from the lung in early disease and promoting edema in late stages of the illness.

Tight Junctions, the Epithelial Barrier, and Toll-like Receptor-4 During Lung Injury.

Lung epithelium is constantly exposed to the environment and is critically important for the orchestration of initial responses to infectious organisms, toxins, and allergic stimuli, and maintenance of normal gaseous exchange and pulmonary function. The integrity of lung epithelium, fluid balance, and transport of molecules is dictated by the tight junctions (TJs). The TJs are formed between adjacent cells. We have focused on the topic of the TJ structure and function in lung epithelial cells. This review includes a summary of the last twenty years of literature reports published on the disrupted TJs and epithelial barrier in various lung conditions and expression and regulation of specific TJ proteins against pathogenic stimuli. We discuss the molecular signaling and crosstalk among signaling pathways that control the TJ structure and function. The Toll-like receptor-4 (TLR4) recognizes the pathogen- and damage-associated molecular patterns released during lung injury and inflammation and coordinates cellular responses. The molecular aspects of TLR4 signaling in the context of TJs or the epithelial barrier are not fully known. We describe the current knowledge and possible networking of the TLR4-signaling with cellular and molecular mechanisms of TJs, lung epithelial barrier function, and resistance to treatment strategies.

Effects of SPA4 peptide on lipopolysaccharide-disrupted lung epithelial barrier, injury, and function in a human cell system and mouse model of lung injury.

Disrupted epithelial barrier, fluid accumulation, inflammation, and compromised physiology are hallmarks of lung injury. Here we investigated the structural stability of the Toll-like receptor-4 (TLR4)-interacting SPA4 peptide, its effect on Pseudomonas aeruginosa lipopolysaccharide (LPS)-disrupted epithelial barrier in a human cell system, and lung injury markers in a mouse model of LPS-induced lung inflammation. The structural properties of SPA4 peptide were investigated using circular dichroism and UV-VIS spectroscopy. The transepithelial electrical resistance (TEER), an indicator of barrier function, was measured after the cells were challenged with 1 µg/ml LPS and treated with 10 or 100 µM SPA4 peptide. The expression and localization of tight junction proteins were studied by immunoblotting and immunocytochemistry, respectively. Mice were intratracheally challenged with 5 µg LPS per g body weight and treated with 50 µg SPA4 peptide. The lung wet/dry weight ratios or edema, surfactant protein-D (SP-D) levels in serum, lung function, tissue injury, body weights, and temperature, and survival were determined as study parameters. The spectroscopy results demonstrated that the structure was maintained among different batches of SPA4 peptide throughout the study. Treatment with 100 µM SPA4 peptide restored the LPS-disrupted epithelial barrier, which correlated with the localization pattern of Zonula Occludens (ZO)-1 and occludin proteins. Correspondingly, SPA4 peptide treatment helped suppress the lung edema and levels of serum SP-D, improved some of the lung function parameters, and reduced the mortality risk against LPS challenge. Our results suggest that the anti-inflammatory activity of the SPA4 peptide facilitates the resolution of lung pathology.

A toll-like receptor-4-interacting surfactant protein-A-derived peptide suppresses tumor necrosis factor-α release from mouse JAWS II dendritic cells.

Surfactant protein-A (SP-A) and Toll-like receptor-4 (TLR4) proteins are recognized as pathogen-recognition receptors. An exaggerated activation of TLR4 induces inflammatory response, whereas SP-A protein down-regulates inflammation. We hypothesized that SP-A-TLR4 interaction may lead to inhibition of inflammation. In this study, we investigated interaction between native baboon lung SP-A and baboon and human TLR4-MD2 proteins by coimmunoprecipitation/immunoblotting and microwell-based methods. The interaction between SP-A and TLR4-MD2 proteins was then analyzed using a bioinformatics approach. In the in silico model of SP-A-TLR4-MD2 complex, we identified potential binding regions and amino acids at the interface of SP-A-TLR4. Using this information, we synthesized a library of human SP-A-derived peptides that contained interacting amino acids. Next, we tested whether the TLR4-interacting SP-A peptides would suppress inflammatory cytokines. The peptides were screened for any changes in the tumor necrosis factor-α (TNF-α) response against lipopolysaccharide (LPS) stimuli in the mouse JAWS II dendritic cell line. Different approaches used in this study suggested binding between SP-A and TLR4-MD2 proteins. In cells pretreated with peptides, three of seven peptides increased TNF-α production against LPS. However, two of these peptides (SPA4: GDFRYSDGTPVNYTNWYRGE and SPA5: YVGLTEGPSPGDFRYSDFTP) decreased the TNF-α production in LPS-challenged JAWS II dendritic cells; SPA4 peptide showed more pronounced inhibitory effect than SPA5 peptide. In conclusion, we identify a human SP-A-derived peptide (SPA4 peptide) that interacts with TLR4-MD2 protein and inhibits the LPS-stimulated release of TNF-α in JAWS II dendritic cells.

Surfactant protein-A and toll-like receptor-4 modulate immune functions of preterm baboon lung dendritic cell precursor cells.

Lung infections are important risk factors for an increased morbidity and mortality in prematurely-delivered babies. Immaturity of the innate immune components makes them extremely susceptible to infection. Recently, we isolated lung dendritic cell (DC)-precursor cells from preterm fetal baboons. The isolated cells were found to be defective in phagocytosing Escherichia coli under basal conditions. In this study, we investigated the effects of exogenously-added purified native lung surfactant protein (SP)-A and recombinant toll-like receptor (TLR)-4-MD2 proteins on phagocytic uptake and cytokine secreting ability of fetal baboon lung DC-precursor cells. The cells were pulsed with SP-A and/or TLR4-MD2 proteins and the phagocytic function was investigated by incubating the cells with fluorescent-labeled E. coli bioparticles and analyzed by spectrofluorometry. The amounts of TNF-α secreted in cell-free supernatants were measured by ELISA. Our results demonstrate that SP-A and TLR4-MD2 proteins, whether added alone or together, induce phagocytosis of E. coli (p<0.05). The SP-A does not affect TNF-α secretion, while the TLR4-MD2 protein induces TNF-α. However, simultaneous addition of SP-A with TLR4-MD2 protein reduces the TLR4-MD2-protein induced TNF-α to basal level. In conclusion, our results indicate that an exogenous administration of SP-A can potentially induce phagocytic activity and anti-inflammatory effect in preterm babies, and help control infection and inflammation.

Mechanism of Anti-Inflammatory Activity of TLR4-Interacting SPA4 Peptide.

The TLR4-interacting SPA4 peptide suppresses inflammation. We assessed the structural and physicochemical properties and binding of SPA4 peptide to TLR4-MD2. We also studied the changes at the whole transcriptome level, cell morphology, viability, secreted cytokines and chemokines, and cell influx in cell systems and mouse models challenged with LPS and treated with SPA4 peptide. Our results demonstrated that the SPA4 peptide did not alter the cell viability and size and only moderately affected the transcriptome of the cells. Computational docking and rendering suggested that the SPA4 peptide intercalates with LPS-induced TLR4-MD2 complex. Results with alanine mutations of D-2 amino acid and NYTXXXRG-12-19 motif of SPA4 peptide suggested their role in binding to TLR4 and in reducing the cytokine response against LPS stimulus. Furthermore, therapeutically administered SPA4 peptide significantly suppressed the secreted levels of cytokines and chemokines in cells and bronchoalveolar lavage fluids of LPS-challenged mice. The results suggest that the SPA4 peptide intercalates with LPS-induced TLR4 complex and signaling for the suppression of inflammation.

Understanding the Host in the Management of Pneumonia. An Official American Thoracic Society Workshop Report.

Pneumonia causes a significant burden of disease worldwide. Although all populations are at risk of pneumonia, those at extremes of age and those with immunosuppressive disorders, underlying respiratory disease, and critical illness are particularly vulnerable. Although clinical practice guidelines addressing the management and treatment of pneumonia exist, few of the supporting studies focus on the crucial contributions of the host in pneumonia pathogenesis and recovery. Such essential considerations include the host risk factors that lead to susceptibility to lung infections; biomarkers reflecting the host response and the means to pursue host-directed pneumonia therapy; systemic effects of pneumonia on the host; and long-term health outcomes after pneumonia. To address these gaps, the Pneumonia Working Group of the Assembly on Pulmonary Infection and Tuberculosis led a workshop held at the American Thoracic Society meeting in May 2018 with overarching objectives to foster attention, stimulate research, and promote funding for short-term and long-term investigations into the host contributions to pneumonia. The workshop involved participants from various disciplines with expertise in lung infection, pneumonia, sepsis, immunocompromised patients, translational biology, data science, genomics, systems biology, and clinical trials. This workshop report summarizes the presentations and discussions and important recommendations for future clinical pneumonia studies. These recommendations include establishing consensus disease and outcome definitions, improved phenotyping, development of clinical study networks, standardized data and biospecimen collection and protocols, and development of innovative trial designs.

In vivo trafficking and immunostimulatory potential of an intranasally-administered primary dendritic cell-based vaccine.

BACKGROUND: Coccidioidomycosis or Valley fever is caused by a highly virulent fungal pathogen: Coccidioides posadasii or immitis. Vaccine development against Coccidioides is of contemporary interest because a large number of relapses and clinical failures are reported with antifungal agents. An efficient Th1 response engenders protection. Thus, we have focused on developing a dendritic cell (DC)-based vaccine for coccidioidomycosis. In this study, we investigated the immunostimulatory characteristics of an intranasal primary DC-vaccine in BALB/c mouse strain that is most susceptible to coccidioidomycosis. The DCs were transfected nonvirally with Coccidioides-Ag2/PRA-cDNA. Expression of DC-markers, Ag2/PRA and cytokines were studied by flow cytometry, dot-immunoblotting and cytometric bead array methods, respectively. The T cell activation was studied by assessing the upregulation of activation markers in a DC-T cell co-culture assay. For trafficking, the DCs were co-transfected with a plasmid DNA encoding HSV1 thymidine kinase (TK) and administered intranasally into syngeneic mice. The trafficking and homing of TK-expressing DCs were monitored with positron emission tomography (PET) using 18F-FIAU probe. Based on the PET-probe accumulation in vaccinated mice, selected tissues were studied for antigen-specific response and T cell phenotypes using ELISPOT and flow cytometry, respectively. RESULTS: We found that the primary DCs transfected with Coccidioides-Ag2/PRA-cDNA were of immature immunophenotype, expressed Ag2/PRA and activated naïve T cells. In PET images and subsequent biodistribution, intranasally-administered DCs were found to migrate in blood, lung and thymus; lymphocytes showed generation of T effector memory cell population (T(EM)) and IFN-γ release. CONCLUSIONS: In conclusion, our results demonstrate that the intranasally-administered primary DC vaccine is capable of inducing Ag2/PRA-specific T cell response. Unique approaches utilized in our study represent an attractive and novel means of producing and evaluating an autologous DC-based vaccine.

Susceptibility of TLR4-defective C3H/HeJ mice to Coccidioides posadasii infection.

Coccidioides posadasii is one of the two fungal pathogens that cause coccidioidomycosis. The inhalation of air-borne arthroconidia leads to the formation of endospore-forming spherules in the lungs and pulmonary infection. In severe condition, the endospores are disseminated to other non-pulmonary organs in the body. The Toll-like receptors (TLR) expressed by a number of immune and non-immune cells can significantly impact the host defense and susceptibility to C. posadasii infection. In this study, we infected TLR4-defective C3H/HeJ mice with a sublethal dose of C. posadasii and studied fungal dissemination, mortality and humoral response. We also measured IL-12 cytokine secreted by C. posadasii-infected dendritic cells. We found that the C3H/HeJ mice were equally susceptible to C. posadasii as compared to C3H/OuJ mice which have intact TLR4. No significant changes were observed in pulmonary fungal load, survival and humoral response. The blockade of TLR4 did not affect C. posadasii-induced IL-12 secretion. However, the fungal counts were 10 times less in spleens of C3H/HeJ mice as compared to C3H/OuJ mice (P<0.05). Our results suggest that the TLR4 may not be involved in inducing protective host defense against C. posadasii, but it appears to be critical for fungal dissemination.

Anti-inflammatory mediators ST2 and SIGIRR are induced by diphenyldifluoroketone EF24 in lipopolysaccharide-stimulated dendritic cells.

The objective of this study was to investigate the effect of EF24, an NF-κB-inhibitor, on the expression of negative regulators in IL-1R pathway, namely ST2 and SIGIRR. Murine JAWS II dendritic cells (DC) were challenged with lipopolysaccharide (LPS, 100 ng/ml) for 4 h, followed by treatment with 10 µM EF24 for 1 h. ST2 and SIGIRR expression was monitored by qRT-PCR and immunoblotting. ST2L and MyD88 interaction was studied by co-immunoprecipitation, and IL-33, a ST2L ligand, was assayed by ELISA. Activation of transcription factor SP1 was examined by confocal microscopy, immunoblotting, and EMSA. The effect of EF24 on accumulation of ubiquitinated proteins in DCs and proteolysis of fluorogenic peptides by purified proteasome was studied. We found that EF24 upregulated the expression of ST2 and SIGIRR and decreased the interaction of the membrane-bound ST2 (ST2L) with MyD88, and significantly reduced IL-33 levels in LPS-stimulated DCs. Simultaneously it increased the activation of transcription factor SP1and restored the basal level of ubiquitinated proteins in LPS-stimulated DCs. Moreover, EF24 inhibited trypsin- and chymotrypsin-like activity of proteasome by directly interacting with 26S proteasome. The results suggest that EF24 activates endogenous anti-inflammatory arm of IL-1R signaling, most likely by stabilizing SP1 against proteasomal degradation.

Dendritic cell-based immunization induces Coccidioides Ag2/PRA-specific immune response.

Valley Fever, or coccidioidomycosis, is caused by a soil-borne, highly virulent fungal pathogen, Coccidioides spp. Infection with Coccidioides can be life-threatening. Since an effective treatment is not available and the T cell-mediated immune response is protective, vaccine development is of interest. In this study, a primary dendritic cell (DC)-vaccine was evaluated for its ability to stimulate Coccidioides antigen-specific immune response in an extremely susceptible BALB/c mouse model. The DC-vaccine (Ag2-DC) was prepared by non-virally transfecting the primary bone marrow-derived DCs with a plasmid DNA encoding Ag2/PRA (protective epitope of Coccidioides). Mice were intranasally immunized with Ag2-DC on days 2 and 10. Immunized mice were necropsied on days 8, 32, and 44. Major organs and blood samples were harvested. The most common indicators of injury (protein, lactate, and albumin), Ag/PRA-specific cytokine-secreting cells, and IgG and its isotypes were determined by biochemical and immunologic assays, respectively. No signs of sickness were noted. Similarly, no significant changes were observed in the levels of total lung protein, lactate, and albumin, in immunized mice compared with healthy control mice. Interferon (IFN-γ), and interleukin (IL)-4 and IL-17 cytokine-secreting cells were observed in lung and lymph nodes upon Ag2-DC immunization. Our results showed that the levels of serum IgG and its isotypes were increased in Ag2-DC-immunized mice. This report provides evidence of DC immunization-stimulated Ag2/PRA-specific immune responses.

TLR4-interacting SPA4 peptide improves host defense and alleviates tissue injury in a mouse model of Pseudomonas aeruginosa lung infection.

Interaction between surfactant protein-A (SP-A) and toll-like receptor (TLR)4 plays a critical role in host defense. In this work, we studied the host defense function of SPA4 peptide (amino acids GDFRYSDGTPVNYTNWYRGE), derived from the TLR4-interacting region of SP-A, against Pseudomonas aeruginosa. We determined the binding of SPA4 peptide to live bacteria, and its direct antibacterial activity against P. aeruginosa. Pro-phagocytic and anti-inflammatory effects were investigated in JAWS II dendritic cells and primary alveolar macrophages. The biological relevance of SPA4 peptide was evaluated in a mouse model of acute lung infection induced by intratracheal challenge with P. aeruginosa. Our results demonstrate that the SPA4 peptide does not interact with or kill P. aeruginosa when cultured outside the host. The SPA4 peptide treatment induces the uptake and localization of bacteria in the phagolysosomes of immune cells. At the same time, the secreted amounts of TNF-α are significantly reduced in cell-free supernatants of SPA4 peptide-treated cells. In cells overexpressing TLR4, the TLR4-induced phagocytic response is maintained, but the levels of TLR4-stimulated TNF-α are reduced. Furthermore, our results demonstrate that the therapeutic administration of SPA4 peptide reduces bacterial burden, inflammatory cytokines and chemokines, intracellular signaling, and lactate levels, and alleviates lung edema and tissue damage in P. aeruginosa-infected mice. Together, our results suggest that the treatment with SPA4 peptide can help control the bacterial burden, inflammation, and tissue injury in a P. aeruginosa lung infection model.

Developmental expression of Toll-like receptors-2 and -4 in preterm baboon lung.

Preterm babies are susceptible to respiratory infection due to immature lung and immune system. Immune cells express Toll-like receptors (TLRs), which may be important in local host defense of preterm infants. We studied the expression of TLR2 and TLR4 in lung tissues of fetal baboons delivered at 125, 140, and 175 days of gestation (dGA; term=185+/-2 days) and preterm baboons that became naturally infected with bacterial/fungal pathogens. The TLR-mRNA and protein were quantified by Northern and Western blotting, respectively. The expression of both TLRs was significantly low at 125 and 140dGA. At 175dGA, the levels reached equivalent to those in adult baboons. However, in naturally infected baboons, the TLR4-mRNA was reduced (p<0.05); TLR2-mRNA expression remained unaltered. The protein expression of both TLRs was found increased in naturally infected baboons. Our results suggest that the lung TLR expression is developmentally regulated and altered during respiratory infection in preterm babies.