Probiotics improve re-epithelialization of scratches infected by Porphyromonas gingivalis through up-regulating CXCL8-CXCR1/CXCR2 axis.

Porphyromonas gingivalis inhibits the release of CXCL8 by gingival epithelial cells and reduces their proliferation. We previously reported that Bifidocaterium sp. and Lactobacillus sp. immunomodulate gingival epithelial cells response to this periodontal pathogen, but their effects on re-epithelialization properties are still unknown. Herein we explored these activities of potential probiotics on gingival epithelial cells and clarified their mechanisms. The immortalized OBA-9 lineage was used to perform in vitro scratches. Twelve clinical isolates and commercially available strains of Bifidobacterium sp. and Lactobacillus sp. were screened. L. casei 324 m and B. pseudolongum 1191A were selected to perform mechanistic assays with P. gingivalis W83 infection and the following parameters were measured: percentage of re-epithelialization by DAPI immunofluorescence area measurement; cell number by Trypan Blue exclusion assay; CXCL8 regulation by ELISA and RT-qPCR; and expression of CXCL8 cognate receptors-CXCR1 and CXCR2 by Flow Cytometry. Complementary mechanistic assays were performed with CXCL8, in the presence or absence of the CXCR1/CXCR2 inhibitor-reparixin. L. casei 324 m and B. pseudolongum 1191A enhanced re-epithelialization/cell proliferation as well as inhibited the harmful effects of P. gingivalis W83 on these activities through an increase in the expression and release of CXCL8 and in the number of cells positive for CXCR1/CXCR2. Further, we revealed that the beneficial effects of these potential probiotics were dependent on activation of the CXCL8-CXCR1/CXCR2 axis. The current findings indicate that these potential probiotics strains may improve wound healing in the context of the periodontal tissues by a CXCL8 dependent mechanism.

The rs1008562, rs2234671, and rs3138060 polymorphisms of the CXCR1 gene are associated with breast cancer risk in a Mexican population.

OBJECTIVE: The rs1008562, rs2234671 and rs3138060 polymorphisms of the CXCR1 gene have been shown to be associated with many diseases, but in breast cancer (BC) their association has not been detected. The purpose of this study was to determine the frequency and association of the rs1008562, rs2234671 and rs3138060 polymorphisms of CXCR1 gene in BC patients in the Mexican population. PATIENTS AND METHODS: The CXCR1 polymorphisms were determined by Polymerase Chain Reaction (PCR) and real time-PCR in healthy Mexican subjects and BC patients. RESULTS: The prevalent patron in BC patients was observed, the majority were overweight and obesity (72%) with metastatic lymph nodes (48%), luminal A/B subtypes (63%), and advanced stages (60%). Triple negative breast cancer (TNBC) patients: they were younger (58%) than 43 years old, overweight (33%), obesity (42%), ductal type histological (98%), metastasis to lymph nodes (47%), advanced stages III-IV (61%) and metastasis (33%). The rs2234671 polymorphism was associated with BC susceptibility when BC patients and the control group were compared for the CC genotype (p=0.037), CG (heterozygous model: p=0.018), GC/CC (dominant model: p=0.004), and the C allele (p=0.001), as well as the GC/CC genotype with hormone replace therapy (HRT, p=0.016). The rs3138060 polymorphism was associated with BC susceptibility for CG/GG genotype (dominant model: p=0.032) and G allele (p=0.018). Although the association between the dominant model of rs1008562, rs2234671, rs3138060 polymorphisms and BC patients and control was evident for tobacco and alcohol consumption (p<0.05). The rs1008562, rs2234671, and rs3138060 polymorphisms of the CXCR1 gene classified by molecular subtype and stage were also associated with BC patients, indicating that these factors may significantly contribute to BC risk. The CCC (OR 1.75, 95% CI 1.03- 2.97, p=0.046), GGG (OR 3.73, 95% CI 1.61- 8.65, p=0.0018) haplotypes were also associated with BC susceptibility. CONCLUSIONS: Rs2234671 and rs3138060 polymorphisms in the CXCR1 gene were associated with BC susceptibility in the Mexican population. The dominant model of the rs1008562, rs2234671 and rs3138060 polymorphisms could significantly contribute to BC risk in tobacco and alcohol consumption, molecular subtype and stage. The rs1008562, rs2234671 and rs3138060 polymorphisms, and the haplotypes CCC and GGG could significantly contribute to BC risk in the Mexican population analyzed.

Single nucleotide polymorphisms in the CXCR1 gene and its association with clinical mastitis incidence in Polish Holstein-Friesian cows.

The aim of this study was to identify the association between single nucleotide polymorphisms (SNPs) in the bovine chemokine receptor (CXCR1) gene and the resistance or susceptibility of cows to mastitis. The analysis of the CXCR1 polymorphism was carried out using polymerase chain reaction restriction fragment length polymorphism analysis for six SNP mutations (c.+291C>T, c.+365T>C, c.+816C>A, c.+819G>A, +1093C>T, and +1373C>A), of which four were located within the coding region and two in the 3'UTR region of the CXCR1 gene. Genetic material from 146 Polish Holstein-Friesian cows was analyzed after dividing into two groups depending on the incidence of clinical mastitis. Identified polymorphisms were in linkage disequilibrium and formed two linkage groups. Three haplotypes (CCCATA, TTAGCC, CTCGCC), forming six haplotype combinations, were detected. The logistic regression showed a significant association between the CC genotype at c.+365T>C and susceptibility of cows to clinical mastitis (P = 0.047). The frequency of haplotype combination 1/1 (CCCATA/CCCATA) was not significantly higher in cows susceptible to mastitis (P = 0.062). Of the identified SNP mutations, only c.+365T>C is a nonsynonymous mutation that induces a change in the coded protein [GCC (Ala) to GTC (Val) at the 122nd amino acid]. This amino acid change can result in changes in receptor function, which may be a reason for the increased mastitis incidence observed in cows with polymorphism at this site.

Tumor Necrosis Factor, but Not Neutrophils, Alters the Metabolic Profile in Acute Experimental Arthritis.

Metabolic alterations are associated with arthritis apart from obesity. However, it is still unclear which is the underlying process behind these metabolic changes. Here, we investigate the role of tumor necrosis factor (TNF) in this process in an acute model of antigen-induced arthritis (AIA). Immunized male BALB/c mice received an intra-articular injection of PBS (control) or methylated bovine serum albumin (mBSA) into their knees, and were also pre-treated with different drugs: Etanercept, an anti-TNF drug, DF2156A, a CXCR1/2 receptor antagonist, or a monoclonal antibody RB6-8C5 to deplete neutrophils. Local challenge with mBSA evoked an acute neutrophil influx into the knee joint, and enhanced the joint nociception, along with a transient systemic metabolic alteration (higher levels of glucose and lipids, and altered adipocytokines). Pre-treatment with the conventional biological Etanercept, an inhibitor of TNF action, ameliorated the nociception and the acute joint inflammation dominated by neutrophils, and markedly improved many of the altered systemic metabolites (glucose and lipids), adipocytokines and PTX3. However, the lessening of metabolic changes was not due to diminished accumulation of neutrophils in the joint by Etanercept. Reduction of neutrophil recruitment by pre-treating AIA mice with DF2156A, or even the depletion of these cells by using RB6-8C5 reduced all of the inflammatory parameters and hypernociception developed after AIA challenge, but could not prevent the metabolic changes. Therefore, the induction of joint inflammation provoked acute metabolic alterations which were involved with TNF. We suggest that the role of TNF in arthritis-associated metabolic changes is not due to local neutrophils, which are the major cells present in this model, but rather due to cytokines.

Methylation analysis of CXCR1 in mammary gland tissue of cows with mastitis induced by Staphylococcus aureus.

Mastitis is the most important disease in the global dairy industry, and causes large economic losses. Staphylococcus aureus is one of most common pathogens that cause bovine mastitis. CXCR1 has been implicated as a prospective genetic marker for mastitis resistance in dairy cows; CXCR1 expression significantly increases when cows have mastitis. To investigate the mechanisms involved in its increased expression, bisulfite sequencing polymerase chain reaction (PCR) was used to detect the methylation status of CXCR1 CpG island, and quantitative fluorescence PCR was used to detect CXCR1 expression in bovine mammary tissue induced with S. aureus in three Chinese Holstein cows. No CpG island was found for bovine CXCR1 in the upstream 2-kb region, whereas one CpG island that contained 13 CpG sites was found in exon 1 of CXCR1. All of the CpG sites were under hypermethylation from 90 to 100% in the mammary tissues. When the mammary gland mRNA expression of CXCR1 was 12.10-fold higher in infected cow quarters than in uninfected quarters, the methylation levels of the CpG site at position 519 were significantly lower in the infected quarters than in the uninfected quarters. Pearson correlation analysis showed that the methylation level at position 519 was significantly negatively correlated with the CXCR1 mRNA expression level (P < 0.05). These results indicate that the methylation of the CpG site at position 519 may regulate CXCR1 expression in cows with mastitis induced by S. aureus, but further studies are needed to elucidate the mechanisms involved.

Association of novel single nucleotide polymorphisms of the CXCR1 gene with the milk performance traits of Chinese native cattle.

Mastitis is an economically devastating disease affecting the dairy industry. Dairy cows with mastitis give reduced milk yield and produce milk that is unfit for consumption. The chemokine receptor CXCR1 is an excellent prospective genetic marker for mastitis resistance in cattle because it regulates neutrophil migration, killing, and survival during infection. We detected 4 single nucleotide polymorphisms (SNPs) of the CXCR1 gene in Chinese native cattle and analyzed their associations with milk traits. Screening for genetic variations in CXCR1 among 648 Chinese Holstein, Luxi Yellow, and Bohai Black cattle by created restriction site polymerase chain reaction (PCR), nested PCR, and DNA sequencing revealed 4 new SNPs with allelic frequencies ranging from 0.676 to 0.821, 0.706 to 0.803, 0.647 to 0.824, and 0.558 to 0.581. All four CXCR1 gene SNPs were located in exon II. Two SNPs, c.337A>G and c.365C>T, were nonsynonymous mutations [ATC (Ile) > GTC (Val) and GCC (Ala) > GTC (Val)], whereas two, c.291C>T and c.333C>T, were synonymous mutations [TTC (Gly) > TTT (Gly) and GGC (Phe) > GGT (Phe)]. Statistical analyses revealed the significant association of c.337A>G and c.365C>T with the somatic cell score, which suggests the possible role of these SNPs in the host response against mastitis. Our data suggest that combined genotypes CCAC/CCGC, CCAC/CTAT, and CCAT/CTAT (lowest somatic cell scores); CTAC/CTAT (highest protein rate); CCAC/CTGC (highest fat rate); and CCAT/CTAT (highest 305-day milk yield) can be used as possible candidates for marker-assisted selection in dairy cattle breeding programs.

Single nucleotide polymorphisms in CXCR1 gene and its association with hepatitis B infected patients in Saudi Arabia.

BACKGROUND/AIM: This study aims to investigate whether the SNPs of CXCR1 gene, could predict the likelihood of viral persistence and/or disease progression. MATERIAL AND METHODS: We investigated the association of two different SNPs (rs2234671, and rs142978743) in 598 normal healthy controls and 662 HBV patients from a Saudi ethnic population. The HBV patients were categorized into inactive carriers (n = 428), active carriers (n = 162), cirrhosis (n = 54) and Cirrhosis-HCC (n = 18) sub-groups. Genetic variants in CXCR1 were determined by polymerase chain reaction (PCR)-based DNA direct sequencing. RESULTS: The frequency of the risk allele 'C' for the SNP, rs2234671 was found to be insignificant when the patient group was compared to the uninfected control group, however, a significant distribution of the allele 'C' of rs2234671 was observed among active HBV carriers + cirrhosis + cirrhosis - HCC vs. inactive HBV carriers with an OR = 1.631 (95% C.I. 1.016-2.616) and p = 0.032. However, no significant association was observed for rs142978743 when the distribution of risk allele was analyzed among the different patient groups (i.e. inactive carriers, active carriers, cirrhosis and HCC). Furthermore, the most common haplotype, Haplo-1 (AG), was found to have an insignificant frequency distribution between HBV cases and controls, while the same haplotype was found to be significantly distributed when active carriers + cirrhosis + cirrhosis - HCC patients were compared to inactive HBV carriers with a frequency of 0.938 and p = 0.0315. Haplo-2 (AC) was also found to be significantly associated with a frequency of 0.058 and p = 0.0163. CONCLUSION: The CXCR1 polymorphism, rs2234671 was found to be associated with chronic HBV infection and may play a role in disease activity.

Variation in the CXCR1 gene (IL8RA) is not associated with susceptibility to chronic periodontitis.

BACKGROUND: The chemokine receptor 1 CXCR-1 (or IL8R-alpha) is a specific receptor for the interleukin 8 (IL-8), which is chemoattractant for neutrophils and has an important role in the inflammatory response. The polymorphism rs2234671 at position Ex2+860G>C of the CXCR1 gene causes a conservative amino acid substitution (S276T). This single nucleotide polymorphism (SNP) seemed to be functional as it was associated with decreased lung cancer risk. Previous studies of our group found association of haplotypes in the IL8 and in the CXCR2 genes with the multifactorial disease chronic periodontitis. In this study we investigated the polymorphism rs2234671 in 395 Brazilian subjects with and without chronic periodontitis. FINDINGS: Similar distribution of the allelic and genotypic frequencies were observed between the groups (p>0.05). CONCLUSIONS: The polymorphism rs2234671 in the CXCR1 gene was not associated with the susceptibility to chronic periodontitis in the studied Brazilian population.

CXCR1 and SLC11A1 polymorphisms affect susceptibility to cutaneous leishmaniasis in Brazil: a case-control and family-based study.

BACKGROUND: L. braziliensis causes cutaneous (CL) and mucosal (ML) leishmaniasis. Wound healing neutrophil (PMN) and macrophage responses made following the bite of the vector sand fly contribute to disease progression in mice. To look at the interplay between PMN and macrophages in disease progression in humans we asked whether polymorphisms at genes that regulate their infiltration or function are associated with different clinical phenotypes. Specifically, CXCR1 (IL8RA) and CXCR2 (IL8RB) are receptors for chemokines that attract PMN to inflammatory sites. They lie 30-260 kb upstream of SLC11A1, a gene known primarily for its role in regulating macrophage activation, resistance to leishmaniasis, and wound healing responses in mice, but also known to be expressed in PMN, macrophages and dendritic cells. METHODS: Polymorphic variants at CXCR1, CXCR2 and SLC11A1 were analysed using Taqman or ABI fragment separation technologies in cases (60 CL; 60 ML), unrelated controls (n = 120), and multicase families (104 nuclear families; 88 ML, 250 CL cases) from Brazil. Logistic regression analysis, family-based association testing (FBAT) and haplotype analysis (TRANSMIT) were performed. RESULTS: Case-control analysis showed association between the common C allele (OR 2.38; 95% CI 1.23-4.57; P = 0.009) of CXCR1_rs2854386 and CL, supported by family-based (FBAT; Z score 2.002; P = 0.045) analysis (104 nuclear families; 88 ML, 250 CL cases). ML associated with the rarer G allele (Z score 1.999; P = 0.046). CL associated with a 3' insertion/deletion polymorphism at SLC11A1 (Z score 2.549; P = 0.011). CONCLUSIONS: The study supports roles for CXCR1 and SLC11A1 in the outcome of L. braziliensis infection in humans. Slc11a1 does not influence cutaneous lesion development following needle injection of Leishmania in mice, suggesting that its role here might relate to the action of PMN, macrophage and/or dendritic cells in the wound healing response to the sand fly bite. Together with the CXCR1 association, the data are consistent with hypotheses relating to the possible role of PMN in initiation of a lesion following the delivery of parasites via the sand fly bite. Association of ML with the rare derived G allele suggests that PMN also have an important positive role to play in preventing this form of the disease.

Treatment with DF 2162, a non-competitive allosteric inhibitor of CXCR1/2, diminishes neutrophil influx and inflammatory hypernociception in mice.

BACKGROUND AND PURPOSE: Neutrophil migration into tissues is involved in the genesis of inflammatory pain. Here, we addressed the hypothesis that the effect of CXC chemokines on CXCR1/2 is important to induce neutrophil migration and inflammatory hypernociception. EXPERIMENTAL APPROACH: Mice were treated with a non-competitive allosteric inhibitor of CXCR1/2, DF 2162, and neutrophil influx and inflammatory hypernociception were assessed by myeloperoxidase assay and electronic pressure meter test, respectively, in various models of inflammation. KEY RESULTS: DF 2162 inhibited neutrophil chemotaxis induced by CXCR1/2 ligands but had no effect on CXCL8 binding to neutrophils. A single mutation of the allosteric site at CXCR1 abrogated the inhibitory effect of DF 2162 on CXCL-8-induced chemotaxis. Treatment with DF 2162 prevented influx of neutrophils and inflammatory hypernociception induced by CXCL1 in a dose-dependent manner. The compound inhibited neutrophil influx and inflammatory hypernociception induced by carrageenan, lipopolysaccharide and zymosan, but not hypernociception induced by dopamine and PGE(2). DF 2162 had a synergistic effect with indomethacin or the absence of TNFR1 to abrogate carrageenan-induced hypernociception. Treatment with DF 2162 diminished neutrophil influx, oedema formation, disease score and hypernociception in collagen-induced arthritis. CONCLUSIONS AND IMPLICATIONS: CXCR1/2 mediates neutrophil migration and is involved in the cascade of events leading to inflammatory hypernociception. In addition to modifying fundamental pathological processes, non-competitive allosteric inhibitors of CXCR1/2 may have the additional benefit of providing partial relief for pain and, hence, may be a valid therapeutic target for further studies aimed at the development of new drugs for the treatment of rheumatoid arthritis.