Cathelicidin Research Articles

A Randomized Controlled Crossover Trial for Reinforcement of Epithelial Barrier Function by Vitamin D Induction of the Antimicrobial Peptide Cathelicidin - A Novel Therapeutic Approach in Chronic Periodontitis

Background: Oral epithelium forms a physical, chemical and immunological barrier against invading microorganisms. The chemical barrier mainly comprises of anti-microbial peptides (AMPs) like cathelicidins. Transcription of cathelicidin anti-microbial peptide (CAMP) gene is induced by binding of the bioactive form of vitamin D to vitamin D receptor. Thus, an increase in vitamin D should significantly improve the chemical barrier function by increasing AMPs. This study explored vitamin D in primary prevention of chronic periodontitis by reinforcing the epithelial barrier by increased action of cathelicidin. This was a single center, randomized, two-way, crossover design study. The aim of this study was to assess the effect of vitamin D administration on levels of cathelicidin LL-37 in gingival crevicular fluid (GCF) of chronic periodontitis patients. Methodology: Forty vitamin D insufficient subjects with chronic periodontitis were divided into two groups. GCF sampling was done a day after the periodontal examination. Following this, all the patients were subjected to scaling and root planning (SRP). Test group patients were prescribed oral dose of 2000 IU vitamin D daily for a period of three months. It was followed by a six week washout period. Then the groups were subjected to crossover and the same treatment sequence was followed. Cathelicidin LL-37 level was estimated using Enzyme Linked Immunosorbent Assay (ELISA) method. Results: Vitamin D administration increased the levels of cathelicidin in periodontal diseases. Cathelicidin levels in GCF increase after vitamin D intake. Conclusion: Periodontal diseases might be prevented by reinforcing the epithelial barrier (chemical barrier) by vitamin D induced production of AMPs.

Liver proteomic analysis reveals acute liver failure induced by lipopolysaccharide/D-galactosamine in rats involved in neutrophil extracellular trap formation

Objectives Acute liver failure (ALF) is a rare life-threatening condition that leads to rapid deterioration of liver function. Although global awareness of ALF consequences is increasing, the precise molecular mechanisms associated with its rapid progression remain unclear. In the present study, we established a rat model of ALF using Lipopolysaccharide (LPS)/D-galactosamine (D-Gal) and explored the potential molecular mechanism of ALF. Methods Multiplexed isobaric tandem mass tag labelling combined with liquid chromatography-mass spectrometry was used to thoroughly screen for differentially expressed proteins in liver samples from LPS/D-Gal-induced ALF rat models. Results We identified 175 proteins, whose expression was altered by at least 1.5-fold, between the liver samples of ALF and control groups. Of these, 14 dysregulated proteins mainly participated in the regulation of neutrophil extracellular trap (NET) formation. Furthermore, rats with severe ALF showed elevated levels of cathelicidin antimicrobial peptide, myeloperoxidase, and fibrinogen gamma chain, consistent with NET formation. These findings suggest that the NET formation pathway may have contributed to the regulation of the clinical features and progression of liver injury in ALF rats. Conclusion To our knowledge, this study is the first to report a global differential protein expression profile of liver samples from rats with LPS/D-Gal-induced ALF. Our TMT-based quantitative proteomic analysis revealed molecular differences involved in NET formation between the ALF and control rat groups, potential therapeutic targets for ALF treatment as well as fundamental information for further detailed experimental research.

Principal Component Analysis to Differentiate Patients with Palmoplantar Pustulosis from Those with Palmoplantar Pustular Psoriasis.

BackgroundPalmoplantar pustulosis (PPP) is initiated from the acrosyringium. However, it is unclear whether PPP should be considered a distinct entity or should be classified into the spectrum of pustular psoriasis, also known as palmoplantar pustular psoriasis (PPPP).ObjectiveWe evaluated the differences in immunohistochemical staining in patients with PPP to determine whether they can be classified into two groups based on psoriatic properties or acrosyringeal properties.MethodsNineteen punch biopsy specimens diagnosed with PPP were collected. Antibodies were chosen for identifying the acrosyringeal properties of α-3-nicotine acetylcholine receptors (α-3-nAChR), psoriatic properties of interleukin (IL)-23 and IL-36R, inflammatory cell properties of human cathelicidin antimicrobial peptide 18/LL-37, IL-8, lipocalin-2 (LCN2), and CD3. The degree of staining of the epidermis was evaluated using the ordinal scale (0~3). The principal component analysis was used to derive principal components (PCs) of common variation between the stains, and the two groups were divided using PCs and cluster analysis.ResultsThree main PCs explained 64% of the total variance in PPP. PC1 (pustular psoriasis properties) showed a higher correlation with IL-36R. PC2 (acrosyringeal/inflammatory properties) showed a higher correlation with α-3-nAChR, IL-8, LCN2, and CD3. PC3 (psoriasis properties) showed a higher correlation with IL-23. PC1 showed a statistically significant difference (p=0.0284) between the two groups. We identified three PCs associated with the pathomechanisms of PPP.ConclusionAlthough PC1 showed a statistically significant difference between the two groups, we did not identify differential protein expression related to the pathogenesis between PPP and PPPP.

Inflammatory response elicited by Ureaplasma parvum colonization in human cervical epithelial, stromal, and immune cells.

Ureaplasma parvum is a commensal bacterium in the female reproductive tract but has been associated with pregnancy complications such as preterm prelabor rupture of membranes and preterm birth (PTB). However, the pathologic effects of U. parvum in the cervix, which prevents ascending infections during pregnancy, are still poorly understood. To determine the impact of U. parvum on the cervix, ectocervical (ecto) and endocervical (endo) epithelial and stromal cells were incubated with U. parvum. Macrophages were also tested as a proxy for cervical macrophages to determine the antigenicity of U. parvum. The effects of U. parvum, including influence on cell cycle and cell death, antimicrobial peptide (AMP) production, epithelial-to-mesenchymal transition (EMT), and inflammatory cytokine levels, were assessed. U. parvum colonized cervical epithelial and stromal cells 4 h post-infection. Like uninfected control, U. parvum neither inhibited cell cycle progression and nor caused cell death in cervical epithelial and stromal cells. U. parvum increased the production of the AMPs cathelicidin and human β-defensin 3 and exhibited weak signs of EMT evidenced by decreased cytokeratin 18 and increased vimentin expression in cervical epithelial cells. U. parvum induced a proinflammatory environment (cytokines) and increased MMP-9 in cervical epithelial cells but promoted pro- and anti-inflammatory response in cervical stromal cells and macrophages. U. parvum may colonize the cervical epithelial layer, but induction of AMPs and anti-inflammatory response may protect the cervix and may prevent ascending infections that can cause PTB. These findings suggest that U. parvum is a weak inducer of inflammation in the cervix.

The anticancer mechanism of human antimicrobial peptide LL-37

Human antimicrobial peptides LL-37 have a variety of medicinal uses. It has been portrayed that this peptide has robust tumoricidal action in a range of malignancies, particularly ovarian cancer, lung cancer, breast cancer, prostate cancer, pancreatic cancer, malignant melanoma, and squamous cell carcinoma of the skin. It exhibits substantial anticancer action against a range of cancers, including colon cancer, gastric cancer, hematologic malignancy, and oral squamous cell carcinoma (OSCC), in comparison. In this review, we explored in depth the anticancer mechanism of action of LL-37 in numerous sorts of cancer. We have shown how LL-37 impedes colon cancer by eliciting caspase-independent apoptosis. LL-37, in addition, has been noticed to boost tumor-suppressive bone morphogenetic protein signaling in gastric cancer cells via restricting the proteasome, which has been previously reported. In this research, we investigated how DNA methylation interferes with the activity of the human CAMP (Cathelicidin antimicrobial peptide gene) promoter and, as a result, acts as a tumor inhibitor in mouth squamous cell carcinoma. Additionally, how LL-37 inhibits cancer cell development in hematologic malignancy has been explored through caspase-independent but Ca2+/calpain- and AIF-dependent processes.

Reinforcement of Oral Epithelial Barrier Function by Vitamin D Induction of the Antimicrobial Peptide Cathelicidin. A Novel Therapeutic Approach in Chronic Periodontitis

Introduction: Oral epithelium forms a physical, chemical and immunological barrier against the invading microorganisms. The chemical barrier mainly comprises of anti-microbial peptides (AMPs) like cathelicidins which are endogenously produced antibiotics. Transcription of the cathelicidin anti-microbial peptide (CAMP) gene is induced by binding of the bioactive form of vitamin D to the vitamin D receptor. Thus, an increase in vitamin D should significantly improve the chemical barrier function by increasing AMPs.
 Through a randomized controlled trial we tend to explore a well accepted molecule like vitamin D in primary prevention of chronic periodontitis by reinforcing the oral epithelial barrier by increased action of cathelicidin.
 Aim of the Study: The aim of this study was to assess the effect of vitamin D administration systemically on levels of cathelicidin LL-37 in gingival crevicular fluid of chronic periodontitis patients.
 Methodology: 40 vitamin D insufficient subjects with chronic periodontitis were divided into 2 groups. Gingival crevicular fluid (GCF) sampling was carried out a day after the periodontal examination. Next all patients were subjected to scaling and root planning (SRP). Test group patients were prescribed oral dose of 2000IU Vitamin D daily for a period of 3months whereas control group was devoid of any medication. GCF sampling was repeated for both the groups after three months. Cathelicidin LL-37 level was estimated using Enzyme Linked Immunosorbent Assay (ELISA)method for both the groups.
 Results: Vitamin D administration increases the levels of cathelicidin in periodontal diseases. Cathelicidin levels in GCF increase after Vitamin D intake.
 Conclusion: Periodontal diseases might be prevented by reinforcing the oral epithelial barrier (chemical barrier) by vitamin D induced production of AMPs.

The adipokine C1q/TNF-related protein-3 (CTRP-3) inhibits Toll-like receptor (TLR)-induced expression of Cathelicidin antimicrobial peptide (CAMP) in adipocytes.

CAMP (Cathelicidin antimicrobial peptide) expression in adipocytes is regulated by Toll-like receptor (TLR) agonists. Secreted adipokines such as CTRP-3 have been suggested to participate in innate immune signaling in adipose tissue (AT). This study investigates whether TLR-induced CAMP expression in adipocytes is antagonized by CTRP-3. 3T3-L1 adipocytes were co-stimulated with TLR agonists (LPS, MALP-2, Pam3CSK4, pI:C) and recombinant CTRP-3. In a SIRS model, C57BL/6 wild-type mice were intraperitoneally (ip) injected with recombinant CTRP-3 prior to LPS. CAMP expression was analyzed by real-time PCR in AT of wild-type mice and in AT and primary adipocytes from transgenic mice lacking adipocyte CTRP-3 expression. Comparative transcriptome analysis by RNA seq. was applied in CTRP-3 KO adipocytes. In vitro, CTRP-3 antagonized TLR4- and TLR1/2-induced CAMP expression in adipocytes whereas TLR3- and TLR2/6-mediated induction of CAMP was not affected. in vivo, application of exogenous CTRP-3 dose-dependently antagonized LPS-induced CAMP expression in intra-abdominal AT. CAMP expression in total AT and in primary adipocytes of subcutaneous and intra-abdominal AT did not differ between wild-type mice and transgenic mice lacking adipocyte CTRP-3 expression. The study suggests a hypothetical role of CAMP in host defense not only against Gram-positive bacteria sensed by TLR1/2 and TLR2/6 but also against Gram-negative bacteria sensed by TLR4 and potentially against viruses sensed by TLR3. The machinery of TLR-mediated pro-inflammatory activation of the CAMP gene in adipocytes seems to be partly modulated by secreted adipokines belonging to the growing family of C1q/TNF-related proteins such as CTRP-3.

MIR337-3p Enhances Mycobacterial Pathogenicity Involving TLR4/MYD88 and STAT3 Signals, Impairing VDR Antimicrobial Response and Fast-Acting Immunity.

Active form of vitamin D (VitD) enhances human innate immunity against Mycobacterium tuberculosis (Mtb) infection. Our previous studies showed that MIR337-3p was highly expressed in lymphocytes of tuberculosis (TB) patients. Here, we identified the mechanism of MIR337-3p in the regulation of fast-acting anti-TB immunity by inhibiting VitD-dependent antimicrobial response pathways. While high-level MIR337-3p expression was induced by mycobacterial infection in cellular models and mice, TB patients exhibited significantly increased MIR337-3p in CD14+ monocytes/macrophages, innate-like Vγ2+ T cells, and CD8+ lymphocytes containing natural killer (NK)/innate lymphoid cells. MIR337-3p promoted the mycobacterial entry/infection and replication/growth in host target cells: macrophages and lung epithelial cells. Such MIR337-3p-enhanced pathogenicity coincided with the MIR337-3p depression of VitD-dependent antimicrobial response of cytochrome P450, family 27, subfamily b, polypeptide 1 (CYP27B1)/Beta-defensin 4 (DEFB4A)/ cathelicidin antimicrobial peptide CAMP pathways. Surprisingly, single MIR337-3p species could specifically target both the Toll-like receptor 4 (TLR4) and signal transducer and activator of transcription 3 (STAT3) 3′-untranslated regions (UTRs) to depress the TLR4/MYD88 and STAT3 signals and impair either of the two signals inhibiting the VitD-dependent antimicrobial pathways in macrophages. Concurrently, human peripheral blood mononuclear cells (PBMCs) expressing high-level MIR337-3p exhibited a reduced ability of innate cell populations to mount fast-acting cellular immunity against intracellular mycobacterial infection. Furthermore, a higher expression of Mir337-3p after mycobacterial infection of mice coincided with much greater colony-forming unit (CFU) counts in lungs and even the death of infected animals, whereas Mir337-3p inhibitor treatment of infected mice reduced Mir337-3p levels and reversed Mir337-3p-mediated increases in CFU counts. Thus, TB-driven single MIR337-3p species could specifically target/impair both TLR4/MYD88 and STAT3 activation signals, inhibiting VitD-dependent antimicrobial response and fast-acting anti-TB immunity, leading to enhanced pathogenicity.

Human Antimicrobial Peptide Triggered Colloidal Transformations in Bacteria Membrane Lipopolysaccharides.

Growing concerns of bacterial resistance against conventional antibiotics shifts the research focus toward antimicrobial peptide (AMP)-based materials. Most AMPs kill gram-negative bacteria by destroying their inner membrane, but have to first pass the outer membrane covered with lipopolysaccharides (LPS). Their interplay with the LPS is crucial for bactericidal activity, but is yet to be elucidated in detail. In this study, self-assemblies of Escherichia coli LPS with the human cathelicidin AMP LL-37, free and encapsulated into glyceryl monooleate (GMO) lipid nanoparticles, are analyzed using synchrotron small angle X-ray scattering, dynamic light scattering, and cryogenic transmission electron microscopy. Circular dichroism spectroscopy is used to study modifications in LL-37's secondary structure. LPS is found to form elongated micelles and the addition of LL-37 induces their transformation to multilamellar structures. LPS' addition to GMO cubosomes triggers the swelling of the internal cubic structure, while in multilamellar GMO/LL-37 nanocarriers it causes transitions into unstructured particles. The insights on the interactions among LPS and LL-37, in its free form or encapsulated in GMO dispersions, may guide the design of LPS-responsive antimicrobial nanocarriers. The findings may further assist the formulation of antimicrobial nanomaterials with enhanced penetration of LPS layers for improved destruction of bacterial membranes.

HIF-1 mediated activation of antimicrobial peptide LL-37 in type 2 diabetic patients

Infections are common in patients with diabetes, but increasing antibiotic resistance hampers successful bacterial clearance and calls for alternative treatment strategies. Hypoxia-inducible factor 1 (HIF-1) is known to influence the innate immune defense and could therefore serve as a possible target. However, the impact of high glucose on HIF-1 has received little attention and merits closer investigation. Here, we show that higher levels of proinflammatory cytokines and CAMP, encoding for the antimicrobial peptide cathelicidin, LL-37, correlate with HIF-1 in type 2 diabetic patients. Chemical activation of HIF-1 further enhanced LL-37, IL-1β, and IL-8 in human uroepithelial cells exposed to high glucose. Moreover, HIF-1 activation of transurethrally infected diabetic mice resulted in lower bacterial load. Drugs activating HIF-1 could therefore in the future potentially have a therapeutic role in clearing bacteria in diabetic patients with infections where antibiotic treatment failed.Key messages• Mohanty et al. “HIF-1 mediated activation of antimicrobial peptide LL-37 in type 2 diabetic patients.”• Our study highlights induction of the antimicrobial peptide, LL-37, and strengthening of the innate immunity through hypoxia-inducible factor 1 (HIF-1) in diabetes.• Our key observations are:1. HIF-1 activation increased LL-37 expression in human urothelial cells treated with high glucose. In line with that, we demonstrated that patients with type 2 diabetes living at high altitude had increased levels of the LL-37.2. HIF-1 activation increased IL-1β and IL-8 in human uroepithelial cells treated with high glucose concentration.3. Pharmacological activation of HIF-1 decreased bacterial load in the urinary bladder of mice with hereditary diabetes.• We conclude that enhancing HIF-1 may along with antibiotics in the future contribute to the treatment in selected patient groups where traditional therapy is not possible.

Dysregulated non-coding telomerase RNA component and associated exonuclease XRN1 in leucocytes from women developing preeclampsia-possible link to enhanced senescence

Senescence in placenta/fetal membranes is a normal phenomenon linked to term parturition. However, excessive senescence which may be induced by telomere attrition, has been associated with preeclampsia (PE). We hypothesized that the telomerase complex in peripheral blood mononuclear cells (PBMC) and circulating telomere associated senescence markers would be dysregulated in women with PE. We measured long non-coding (nc) RNA telomerase RNA component (TERC) and RNAs involved in the maturation of TERC in PBMC, and the expression of TERC and 5′–3′ Exoribonuclease 1 (XRN1) in extracellular vesicles at 22–24 weeks, 36–38 weeks and, 5-year follow-up in controls and PE. We also measured telomere length at 22–24 weeks and 5-year follow-up. The circulating senescence markers cathelicidin antimicrobial peptide (CAMP), β-galactosidase, stathmin 1 (STMN1) and chitotriosidase/CHIT1 were measured at 14–16, 22–24, 36–38 weeks and at 5-year follow-up in the STORK study and before delivery and 6 months post-partum in the ACUTE PE study. We found decreased expression of TERC in PBMC early in pregnant women who subsequently developed PE. XRN1 involved in the maturation of TERC was also reduced in pregnancy and 5-year follow-up. Further, we found that the senescence markers CAMP and β-galactosidase were increased in PE pregnancies, and CAMP remained higher at 5-year follow-up. β-galactosidase was associated with atherogenic lipid ratios during pregnancy and at 5-year follow-up, in PE particularly. This study suggests a potential involvement of dysfunctional telomerase biology in the pathophysiology of PE, which is not restricted to the placenta.

Antimicrobial peptide gene expression in Atlantic salmon (Salmo salar) seven days post-challenge with Neoparamoeba perurans

Amoebic gill disease in teleost fish is caused by the marine parasite Neoparamoeba perurans. To date, the role of antimicrobial peptides β-defensins and cathelicidins in this infection have not been explored. Using a high-throughput microfluidics quantitative polymerase chain reaction system (Biomark HD™ by Fluidigm), this study aimed to: firstly, to investigate organ-specific expression of antimicrobial peptide genes β-defensin-1, -3 and -4 and cathelicidin 2 in healthy Atlantic salmon; secondly, to compare the expression of these antimicrobial peptide genes in healthy versus asymptomatic Atlantic salmon seven days post-challenge with Neoparamoeba perurans.Results from this study indicate expression of the β-defensin and cathelicidin genes in the selected organs from healthy Atlantic salmon. Furthermore, a statistically significant upregulation of β-defensins −3 and −4 and cathelicidin 2 was detected in gill of parasite-challenged salmon. The upregulated cathelicidin and β-defensin genes in gill could indicate novel potential roles in innate immune responses to Neoparamoeba perurans.

217 Tofacitinib suppresses IL-10/IL-10R signaling and modulates host defense responses in human macrophages

JAK inhibitors are increasingly used in dermatology. Despite broad inhibitory effects on cytokine signaling cascades, they only modestly increase the risk for infectious diseases. To address molecular mechanisms underlying this unexpected clinical observation, we investigated how tofacintib, a first-in-class JAK inhibitor, regulates host defense responses in TLR4-activated human macrophages. Specifically, we asked if tofacitinib inhibits anti-inflammatory IL-10 signaling, thereby counteracting downregulation of inflammatory, host-protective pathways. We found that tofacitinib blocked macrophage responses to IL-10 at the level of STAT3 phosphorylation. Furthermore, TLR4-induced, auto-/paracrine IL-10/IL-10R activation promoted expression of hepcidin, the master regulator of iron metabolism, resulting in intracellular iron sequestration. In contrast, auto-/paracrine IL-10/IL-10R activation repressed expression of cathelicidin antimicrobial peptide, as well as antigen-presenting molecules, thus together, inducing a pathogen-favoring environment. While tofacitinib further repressed cathelicidin, it prevented induction of intracellular hepcidin, and restored expression of antigen-presentation molecules in TLR4-activated macrophages. Our study supports the concept that induction of IL-10/IL-10R signaling drives a complex immune evasion strategy of intracellular microbes. Moreover, we conclude that tofacitinib has diverging effects on macrophage host response pathways, and we identify the TLR4-IL-10-STAT3-hepcidin axis as a potential therapeutic target to counteract immune evasion.

Metformin strengthens uroepithelial immunity against E. coli infection

Urinary tract infection frequently caused by E. coli is one of the most common bacterial infections. Increasing antibiotic resistance jeopardizes successful treatment and alternative treatment strategies are therefore mandatory. Metformin, an oral antidiabetic drug, has been shown to activate macrophages in the protection against certain infecting microorganisms. Since epithelial cells often form the first line of defense, we here investigated the effect on uroepithelial cells during E. coli infection. Metformin upregulated the human antimicrobial peptides cathelicidin LL-37 and RNase7 via modulation of the TRPA1 channel and AMPK pathway. Interestingly, metformin stimulation enriched both LL-37 and TRPA1 in lysosomes. In addition, metformin specifically increased nitric oxide and mitochondrial, but not cytosolic ROS. Moreover, metformin also triggered mRNA expression of the proinflammatory cytokines IL1B, CXCL8 and growth factor GDF15 in human uroepithelial cells. The GDF15 peptide stimulated macrophages increased LL-37 expression, with increased bacterial killing. In conclusion, metformin stimulation strengthened the innate immunity of uroepithelial cells inducing enhanced extracellular and intracellular bacterial killing suggesting a favorable role of metformin in the host defense.

Identification of novel biomarkers of inflammation in Atlantic salmon (Salmo salar L.) by a plasma proteomic approach

Monitoring fish welfare has become a central issue for the fast-growing aquaculture industry, and finding proper biomarkers of stress, inflammation and infection is necessary for surveillance and documentation of fish health. In this study, a proteomic approach using mass spectrometry was applied to identify indicators of the acute response in Atlantic salmon blood plasma by comparing Aeromonas salmonicida subsp. salmonicida infected fish and non-infected controls. The antimicrobial proteins cathelicidin (CATH), L-plastin (Plastin-2, LCP1) and soluble toll-like receptor 5 (sTLR5) were uniquely or mainly identified in the plasma of infected fish. In addition, five immune-related proteins showed significantly increased expression in plasma of infected fish: haptoglobin, high affinity immunoglobulin Fc gamma receptor I (FcγR1, CD64), leucine-rich alpha 2 glycoprotein (LRG1), complement C4 (C4) and phospholipase A2 inhibitor 31 kDa subunit-like protein. However, various fibrinogen components, CD209 and CD44 antigen-like molecules decreased in infected fish. Selected biomarkers were further verified by Western blot analysis of plasma and real time PCR of spleen and liver, including CATH1, CATH2 and L-plastin. A significant increase of L-plastin occurred as early as 24 h after infection, and a CATH2 increase was observed from 72 h in plasma of infected fish. Real time PCR of selected genes confirmed increased transcription of CATH1 and CATH2. In addition, serum amyloid A mRNA significantly increased in liver and spleen after bacterial infection. However, transcription of L-plastin was not consistently induced in liver and spleen. The results of the present study reveal novel and promising biomarkers of the acute phase response and inflammation in Atlantic salmon.

Tofacitinib Suppresses IL-10/IL-10R Signaling and Modulates Host Defense Responses in Human Macrophages

Jak inhibitors are increasingly used in dermatology. Despite broad inhibitory effects on cytokine signaling cascades, they only modestly increase the risk for infectious diseases. To address the molecular mechanisms underlying this unexpected clinical observation, we investigated how tofacintib (tofa), a first-in-class Jak inhibitor, regulates host defense responses in toll-like receptor 4-activated human macrophages. Specifically, we asked whether tofa inhibits anti-inflammatory IL-10 signaling, thereby counteracting the downregulation of inflammatory, host-protective pathways. We found that tofa blocked macrophage responses to IL-10 at the level of signal transducer and activator of transcription 3 phosphorylation. Furthermore, toll-like receptor 4-induced, autocrine/paracrine IL-10/IL-10R activation promoted the expression of hepcidin, the master regulator of iron metabolism, resulting in intracellular iron sequestration. In contrast, autocrine/paracrine IL-10/IL-10R activation repressed the expression of cathelicidin antimicrobial peptide as well as antigen-presenting molecules, thus together, inducing a pathogen-favoring environment. Although tofa further repressed cathelicidin, it prevented the induction of intracellular HAMP and restored the expression of antigen-presentation molecules in toll-like receptor 4-activated macrophages. Our study supports the concept that induction of IL-10/IL-10R signaling drives a complex immune evasion strategy of intracellular microbes. Moreover, we conclude that tofa has diverging effects on macrophage host response pathways, and we identify the toll-like receptor 4-IL-10-signal transducer and activator of transcription 3-HAMP axis as a potential therapeutic target to counteract immune evasion.

Characteristics and Significance of Gene Expression Changes in Peripheral Blood of Lumbar Disc Extrusion Patients before and after Nonoperative Treatment

To define the gene expression characteristics in the peripheral blood of patients with lumbar disc extrusion (LDE) and the effect of nonoperative treatment on the gene expression. DNA microarray was used to identify semi-quantitatively the differentially expressed genes (DEGs) in the peripheral blood of patients with LDE and that of the healthy controls and the variation trend of these DEGs after nonoperative treatment. Enrichment analysis was done to reveal the functional characteristics of these DEGs, and network analysis was done to identify key genes that contribute to gene dysregulation. The levels of these key genes were measured by qRT-PCR to examine their expression in LDE patients and the controls, and the effect of nonoperative treatment on the expression level. We identified 153 DEGs in the peripheral blood of LDE patients and healthy controls, including 131 upregulated genes and 22 downregulated genes. Enrichment analysis revealed that most of the DEGs were related to immunity and the inflammatory response. Network analysis revealed that toll-like receptor 4 ( TLR4 ), matrix metallopeptidase 9 ( MMP9) and myeloperoxidase ( MPO), cathelicidin antimicrobial peptide ( CAMP), resistin ( RETN), toll-like receptor 5 ( TLR5) were the key genes in the protein-protein interaction network. These key genes were all enriched into the terms releated to immunity and the inflammatory response. The patients experienced pain relief after nonoperative treatment. Among the 153 DEGs, TLR5 , interleukin 1 receptor antagonist ( IL1 RN) and solute carrier family 8 member A1 ( SLC8 A1) were downregulated after nonoperative treatment. qRT-PCR revealed that the levels of TLR4, MMP9 , MPO, CAMP, RETN, TLR5, IL1 RN and SLC8 A1 in the peripheral blood of the LDE patients were higher than those of the healthy control group ( P<0.05). In addition, TLR5 , IL1 RN and SLC8 A1 expression levels decreased after treatmentin in comparison with the levels before treatment ( P<0.05). Gene expression in the peripheral blood of LDE patients was characterized by the dysregulation of immune and inflammatory response-related genes, among which, TLR4, MMP9, MPO, CAMP, RETN and TLR5, the genes relevant to immune and inflammatory response, played a key role in the dysregulation of gene expression in the peripheral blood of LDE patients. The outcome of non-operative treatment may be related to the downregulation of the overexpressed TLR5, IL1 RN and SLC8 A1 in the peripheral blood of patients.

Protectins PCTR1 and PD1 Reduce Viral Load and Lung Inflammation During Respiratory Syncytial Virus Infection in Mice.

Viral pneumonias are a major cause of morbidity and mortality, owing in part to dysregulated excessive lung inflammation, and therapies to modulate host responses to viral lung injury are urgently needed. Protectin conjugates in tissue regeneration 1 (PCTR1) and protectin D1 (PD1) are specialized pro-resolving mediators (SPMs) whose roles in viral pneumonia are of interest. In a mouse model of Respiratory Syncytial Virus (RSV) pneumonia, intranasal PCTR1 and PD1 each decreased RSV genomic viral load in lung tissue when given after RSV infection. Concurrent with enhanced viral clearance, PCTR1 administration post-infection, decreased eosinophils, neutrophils, and NK cells, including NKG2D+ activated NK cells, in the lung. Intranasal PD1 administration post-infection decreased lung eosinophils and Il-13 expression. PCTR1 increased lung expression of cathelicidin anti-microbial peptide and decreased interferon-gamma production by lung CD4+ T cells. PCTR1 and PD1 each increased interferon-lambda expression in human bronchial epithelial cells in vitro and attenuated RSV-induced suppression of interferon-lambda in mouse lung in vivo. Liquid chromatography coupled with tandem mass spectrometry of RSV-infected and untreated mouse lungs demonstrated endogenous PCTR1 and PD1 that decreased early in the time course while cysteinyl-leukotrienes (cys-LTs) increased during early infection. As RSV infection resolved, PCTR1 and PD1 increased and cys-LTs decreased to pre-infection levels. Together, these results indicate that PCTR1 and PD1 are each regulated during RSV pneumonia, with overlapping and distinct mechanisms for PCTR1 and PD1 during the resolution of viral infection and its associated inflammation.

Anti-Inflammatory and Anti-Allergic Effects of Saponarin and Its Impact on Signaling Pathways of RAW 264.7, RBL-2H3, and HaCaT Cells.

Saponarin{5-hydroxy-2-(4-hydroxyphenyl)-6-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]-7-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one}, a flavone found in young green barley leaves, is known to possess antioxidant, antidiabetic, and hepatoprotective effects. In the present study, the anti-inflammatory, anti-allergic, and skin-protective effects of saponarin were investigated to evaluate its usefulness as a functional ingredient in cosmetics. In lipopolysaccharide-induced RAW264.7 (murine macrophage) cells, saponarin (80 μM) significantly inhibited cytokine expression, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, inducible nitric oxide synthase, and cyclooxygenase (COX)-2. Saponarin (80 μM) also inhibited the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 involved in the mitogen-activated protein kinase signaling pathway in RAW264.7 cells. Saponarin (40 μM) significantly inhibited β-hexosaminidase degranulation as well as the phosphorylation of signaling effectors (Syk, phospholipase Cγ1, ERK, JNK, and p38) and the expression of inflammatory mediators (tumor necrosis factor [TNF]-α, IL-4, IL-5, IL-6, IL-13, COX-2, and FcεRIα/γ) in DNP-IgE- and DNP-BSA-stimulated RBL-2H3 (rat basophilic leukemia) cells. In addition, saponarin (100 μM) significantly inhibited the expression of macrophage-derived chemokine, thymus and activation-regulated chemokine, IL-33, thymic stromal lymphopoietin, and the phosphorylation of signaling molecules (ERK, p38 and signal transducer and activator of transcription 1 [STAT1]) in TNF-α- and interferon (IFN)-γ-stimulated HaCaT (human immortalized keratinocyte) cells. Saponarin (100 μM) also significantly induced the expression of hyaluronan synthase-3, aquaporin 3, and cathelicidin antimicrobial peptide (LL-37) in HaCaT cells, which play an important role as skin barriers. Saponarin remarkably inhibited the essential factors involved in the inflammatory and allergic responses of RAW264.7, RBL-2H3, and HaCaT cells, and induced the expression of factors that function as physical and chemical skin barriers in HaCaT cells. Therefore, saponarin could potentially be used to prevent and relieve immune-related skin diseases, including atopic dermatitis.

RELATION BETWEEN BACTERIAL COLONIZATION IN LUNGS AND LEVELS OF 25-HYDROXYVITAMIN D AND ANTIMICROBIAL PEPTIDE LL-37 IN CHILDREN WITH CYSTIC FIBROSIS

The aim of the study was to assess the bacterial colonization in lungs of children with cystic fibrosis based on the antimicrobial peptide cathelicidin and 25-hydroxycholecalciferol in the serum. Study showed significant correlation between P. aeruginosa infection and cathelicidin and 25-hydroxycholecalciferol levels.