The Association of HLA-G Gene Polymorphism and Its Soluble Form With Male Infertility

Successful reproduction depends on many factors. Male factors contribute to infertility in approximately 50% of couples who fail to conceive. Seminal plasma consists of secretions from different accessory glands containing a mixture of various cytokines, chemokines, and growth factors, which together can induce a local immune response that might impact on a male’s as well as a female’s fertility. Human leukocyte antigen (HLA)-G expression has been suggested as an immunomodulatory molecule that influences pregnancy outcome. The HLA-G gene encodes either membrane-bound or/and soluble proteins. The aim of this study was the evaluation of HLA-G polymorphisms and their impact on soluble HLA-G (sHLA-G) production. We tested the HLA-G polymorphism in three positions: rs1632947: c.-964G>A; rs1233334: c.-725G>C/T in the promoter region; rs371194629: c.∗65_∗66insATTTGTTCATGCCT in the 3′ untranslated region. We tested two cohorts of men: 663 who participated in in vitro fertilization (test material was blood or sperm), and 320 fertile controls who possessed children born after natural conception (test material was blood). Since 50% of men visiting assisted reproductive clinics have abnormal semen parameters, we wondered if men with normal sperm parameters differ from those with abnormal parameters in terms of HLA-G polymorphism and secretion of sHLA-G into semen. We found that certain rs1632947-rs1233334-rs371194629 HLA-G haplotypes and diplotypes were associated with male infertility, while others were protective. Normozoospermic men with the A-C-del haplotype and A-C-del/A-C-del diplotype secreted the most sHLA-G into semen (574.1 IU/mL and 1047.0 IU/mL, respectively), while those with the G-C-ins haplotype and G-C-ins/G-C-ins diplotype – the least (80.8 IU/mL and 75.7 IU/mL, respectively). Men with the remaining haplotypes/diplotypes secreted sHLA-G at an intermediate level. However, only in one haplotype, namely G-C-ins, did we observe strong significant differences in the concentration of sHLA-G in the semen of men with teratozoospermia compared to men with normal sperm parameters (p = 0.009). In conclusion, fertile men differ in the profile of HLA-G polymorphism from men participating in IVF. Among all HLA-G haplotypes, the most unfavorable for male fertility is the G-C-ins haplotype, which determines the secretion of the lowest concentration of the soluble HLA-G molecule. This haplotype may reduce sperm parameters.

Systematic Evaluation of HLA-G 3’Untranslated Region Variants in Locally Advanced, Non-Metastatic Breast Cancer Patients: UTR-1, 2 or UTR-4 are Predictors for Therapy and Disease Outcome

Despite major improvements in diagnostics and therapy in early as well as in locally advanced breast cancer (LABC), metastatic relapse occurs in about 20% of patients, often explained by early micro-metastatic spread into bone marrow by disseminated tumor cells (DTC). Although neoadjuvant chemotherapy (NACT) has been a successful tool to improve overall survival (OS), there is growing evidence that various environmental factors like the non-classical human leukocyte antigen-G (HLA-G) promotes cancer invasiveness and metastatic progression. HLA-G expression is associated with regulatory elements targeting certain single-nucleotide polymorphisms (SNP) in the HLA-G 3’ untranslated region (UTR), which arrange as haplotypes. Here, we systematically evaluated the impact of HLA-G 3’UTR polymorphisms on disease status, on the presence of DTC, on soluble HLA-G levels, and on therapy and disease outcome in non-metastatic LABC patients. Although haplotype frequencies were similar in patients (n = 142) and controls (n = 204), univariate analysis revealed that the UTR-7 haplotype was related to patients with low tumor burden, whereas UTR-4 was associated with tumor sizes >T1. Furthermore, UTR-4 was associated with the presence of DTC, but UTR-3 and UTR-7 were related to absence of DTC. Additionally, increased levels of soluble HLA-G molecules were found in patients carrying UTR-7. Regarding therapy and disease outcome, univariate and multivariate analysis highlighted UTR-1 or UTR-2 as a prognostic parameter indicative for a beneficial course of disease in terms of complete response towards NACT or progression-free survival (PFS). At variance, UTR-4 was an independent risk factor for a reduced OS besides already known parameters. Taken into account the most common HLA-G 3’UTR haplotypes (UTR-1–UTR-7, UTR-18), deduction of the UTR-1/2/4 haplotypes to specific SNPs revealed that the +3003C variant, unique for UTR-4, seemed to favor a detrimental disease outcome, while the +3187G and +3196G variants, unique for UTR-1 or UTR-2, were prognostic parameters for a beneficial course of disease. In conclusion, these data suggest that the HLA-G 3’UTR variants +3003C, +3187G, and +3196G are promising candidates for the prediction of therapy and disease outcome in LABC patients.

HLA-G in Allergy: Does It Play an Immunoregulatory Role?

Allergy is an inflammatory process determined by a cascade of immune events characterized by T-helper 2 lymphocytes polarization leading to interleukin-4 upregulation, IgE secretion, and mast cell and eosinophil activation. HLA-G molecules, both in membrane-bound and in soluble forms, are known to play a key immunoregulatory role and their involvement in allergic diseases is supported by increasing literature data. HLA-G expression and secretion is specifically induced in peripheral blood mononuclear cells of allergic patients after in vitro incubation with the causal allergen. Elevated levels of soluble HLA-G molecules are detected in serum of patients with allergic rhinitis correlating with allergen-specific IgE levels, clinical severity, drug consumption and response to allergen-specific immunotherapy. HLA-G genetic polymorphisms confer susceptibility to allergic asthma development and high levels of soluble HLA-G molecules are found in plasma and bronchoalveolar lavage fluid of patients with allergic asthma correlating with allergen-specific IgE levels. Interestingly, allergic pregnant women have lower plasma sHLA-G levels than non-allergic women during the 3rd trimester of pregnancy and at delivery. Finally, in allergic patients with atopic dermatitis HLA-G molecules are expressed by T cells, monocytes-macrophages and Langerhans cells infiltrating the dermis. Although at present is difficult to completely define the role of HLA-G molecules in allergic diseases, it may be suggested that they are specifically expressed and secreted by immune cells during the allergic reaction in an attempt to suppress allergic inflammation.

HLA-G 14bp ins/del Polymorphism, Plasma Level of Soluble HLA-G and Association with IL-6/IL-10 Ratio and Survival of Glioma Patients

HLA-G is an immune checkpoint molecule with immunosuppressive and anti-inflammatory activities, and its expression and level of its soluble form (sHLA-G) may play an important role in tumor prognosis. The HLA-G 14 bp ins/del polymorphism and the plasma level of soluble HLA-G (sHLA-G) were investigated by a polymerase chain reaction and ELISA, respectively, in 59 glioma patients. A significantly higher proportion of glioma patients had the 14 nt insert in both homozygous and heterozygous states compared to the control group. Glioma patients had also higher plasma levels of sHLA-G. Patients with methylated MGMT promoter had lower levels of sHLA-G than those with unmethylated MGMT promoter. Level of sHLA-G negatively correlated with the overall survival of patients. Glioblastoma patients who survived more than one year after diagnosis had lower levels of sHLA-G than those surviving less than one year. Patients with sHLA-G levels below the cut off value 40 U/mL survived significantly longer than patients with sHLA-G above 40 U/mL. The levels of sHLA-G also negatively correlated with the level of IL-6 (P=0.0004) and positively with IL-10/IL-6 (P=0.046). Conclusion: The presence of 14 nt insert in both homozygous and heterozygous states of the HLA-G 14 bp ins/del polymorphism is more frequent in glioma patients and the elevated plasma levels of sHLA-G are negatively associated with their survival.

Decreased Neurofilament L Chain Levels in Cerebrospinal Fluid and Tolerogenic Plasmacytoid Dendritic Cells in Natalizumab-Treated Multiple Sclerosis Patients – Brief Research Report

BackgroundNeurofilament Light (NfL) chain levels in both cerebrospinal fluid (CSF) and serum have been correlated with the reduction of axonal damage in multiple sclerosis (MS) patients treated with Natalizumab (NTZ). However, little is known about the function of plasmacytoid cells in NTZ-treated MS patients.ObjectiveTo evaluate CSF NfL, serum levels of soluble-HLA-G (sHLA-G), and eventual tolerogenic behavior of plasmacytoid dendritic cells (pDCs) in MS patients during NTZ treatment.MethodsCSF NfL and serum sHLA-G levels were measured using an ELISA assay, while pDCs (BDCA-2+) were accessed through flow cytometry analyses.ResultsCSF levels of NfL were significantly reduced during NTZ treatment, while the serum levels of sHLA-G were increased. Moreover, NTZ treatment enhanced tolerogenic (HLA-G+, CD274+, and HLA-DR+) molecules and migratory (CCR7+) functions of pDCs in the peripheral blood.ConclusionThese findings suggest that NTZ stimulates the production of molecules with immunoregulatory function such as HLA-G and CD274 programmed death-ligand 1 (PD-L1) which may contribute to the reduction of axonal damage represented by the decrease of NfL levels in patients with MS.

Genetic polymorphism of HLA-G gene (G*01:03, G*01:04, and G*01:05N) in Iraqi patients with inflammatory bowel disease (ulcerative colitis and Crohn’s disease)

Background Human leukocyte antigen-G (HLA-G) has been proposed to influence susceptibility to inflammatory bowel disease (IBD). Therefore, the genetic association between HLA-G alleles and two clinical phenotypes of IBD (ulcerative colitis [UC] and Crohn’s disease [CD]) was evaluated in Iraqi patients. A case-control study was performed on 50 UC and 50 CD patients and 100 healthy controls (HC). Three HLA-G alleles (G*01:03, G*01:04, and G*01:05N) were determined using sequence-specific polymerase chain reaction assay followed by product digestion with restriction endonucleases (Hinf-I, BseR-I, and PpuM-I, respectively). Results The G*01:03 allele was not detected in IBD patients (UC and CD) or HC, while G*01:04 and G*01:05N alleles showed polymorphic frequencies. The allele G*01:04 was significantly associated with susceptibility to UC (odds ratio [OR] = 2.55; 95% confidence interval [CI] = 1.27–5.13; corrected probability [pc] = 0.018) and CD (OR = 4.45; 95% CI = 2.11–9.41; pc < 0.001). The allele G*01:05N was also associated with increased risk of UC (OR = 4.17; 95% CI = 1.32–13.21; pc = 0.032) and CD (OR = 4.75; 95% CI = 1.53–14.78; pc = 0.014). These associations were more pronounced in IBD (UC + CD), and a significantly increased risk for IBD was found with the alleles G*01:04 (OR = 3.32; 95% CI = 1.86–5.95; pc < 0.001) and G*01:05N (OR = 4.46; 95% CI = 1.59–12.47; pc = 0.008). A stratification of IBD patients according to some demographic and clinical characteristics revealed that frequencies of both alleles showed no significant differences between the subgroups of patients in each stratum. Soluble HLA-G was not influenced by HLA-G alleles in patients or HC. UC was an exception, and the presence of G*01:04 allele was associated with a significantly higher mean of soluble HLA-G compared to patients without the allele (189.6 ± 24.0 vs. 168.6 ± 27.2 ng/mL; p = 0.033). Conclusion This study indicated that HLA-G*01:04 and HLA-G*01:05N alleles may influence susceptibility to UC and CD in Iraqi patients.

Cytokines and Soluble HLA-G Levels in the Acute and Recovery Phases of Arbovirus-Infected Brazilian Patients Exhibiting Neurological Complications

Severe neurological complications following arbovirus infections have been a major concern in seasonal outbreaks, as reported in the Northeast region of Brazil, where the same mosquito transmitted Zika (ZIKV), Dengue (DENV), and Chikungunya (CHIKV) viruses. In this study, we evaluated the levels of 36 soluble markers, including cytokines, chemokines, growth factors, and soluble HLA-G (Luminex and ELISA) in: i) serum and cerebrospinal fluid (CSF), during the acute phase and two years after the infection (recovery phase, only serum), ii) the relationship among all soluble molecules in serum and CSF, and iii) serum of infected patients without neurological complications, during the acute infection. Ten markers (sHLA-G, IL-10, IL-22, IL-8, MIP-1α, MIP-1β, MCP-1, HGF, VEGF, and IL-1RA) exhibited differential levels between the acute and recovery phases, with pronounced increases in MIP-1α (P&lt;0.0001), MCP-1 (P&lt;0.0001), HGF (P= 0.0001), and VEGF (P&lt;0.0001) in the acute phase. Fourteen molecules (IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-13, IL-15, IL-17A, IFN-α, TNF, and G-CSF) exhibited distinct levels between arbovirus patients presenting or not neurological complications. IL-8, EGF, IL-6, and MCP-1 levels were increased in CSF, while RANTES and Eotaxin levels were higher in serum. Soluble serum (IL-22, RANTES, Eotaxin) and CSF (IL-8, EGF, IL-3) mediators may discriminate putative risks for neurological complications following arbovirus infections. Neurological complications were associated with the presence of a predominant inflammatory profile, whereas in non-complicated patients an anti-inflammatory profile may predominate. Mediators associated with neuroregeneration (EGF and IL-3) may be induced in response to neurological damage. Broad spectrum immune checkpoint molecules (sHLA-G) interact with cytokines, chemokines, and growth factors. The identification of soluble markers may be useful to monitor neurological complications and may aid in the development of novel therapies against neuroinflammation.