Dissecting toll-like receptor molecular regulatory network(s) in reproductive medicine, primarily infertility: A snapshot

Overview of TLR signal transduction

The identification of similarity between the cytoplasmic domains of Drosophila Toll and mammalian Interleukin (IL)-1 receptor propelled the search for orthologous receptors, subsequently leading to the discovery of “human Toll” (later named TLR4) in humans.^[9] The immunobiology of TLRs is indeed complex; TLRs are type 1 integral membrane glycoproteins comprising leucine-rich repeat (LRR) motifs in the pathogen-binding ectodomains (ECD), and cytoplasmic signaling domains known as Toll IL-1 receptor (TIR) domains, joined by a single transmembrane helix.^[11] TLRs are expressed in diverse cell types (cervical, airway, gut epithelial cells, B cells, mast cells, NK cells, dendritic cells, regulatory T cells, macrophages, monocytes, neutrophils, basophils, and endothelial cells); few TLRs (1, 2, 4, 5, and 6) are expressed on the cell surface while other TLRs (3, 7, 8 and 9) are localized intracellularly.^[8,9,11] TLRs can recognize a variety of pathogen-associated molecular patterns (PAMPs), including lipoprotein, lipopolysaccharide (LPS), peptidoglycan, zymosan, bacterial flagella, CpG deoxyribonucleic acid (DNA), and double strand and single strand ribonucleic acids (RNAs). Moreover, subcellular distribution of TLRs may be defined by their transmembrane and/or cytoplasmic domains.^[12] Furthermore, deciphering the complexities associated with TLR signaling network(s) has been one of the primary objectives of molecular biology research-oriented experimental studies in recent years. TLR signal transduction involves myeloid differentiation factor (MyD)88-mediated pathway that leads to the activation of nuclear factor (NF)-kB and TIR domain-containing adaptor inducing Interferon (IFN)-beta (TRIF)-mediated pathway resulting in the activation of interferon regulatory factor (IRF)-3.^[8] Two adaptor proteins, namely, the MyD88 and TRIF, also known as TIR-containing adaptor molecule, are the critical “molecular regulatory switches” of the TLR signal transduction pathway(s). MyD88 recruits members of the IL-1R-associated kinase (IRAK) family (IRAK-1 and IRAK-4) and TNF receptor-associated factor (TRAF-6).^[8] Linker molecules, transforming growth factor beta (TGF-beta)-activated kinase (TAK-1), TAK-binding protein (TAB)-1, and TAB-2 activate downstream IkB kinase kinases (IKK); in a complex series of sequential cascade of subsequent signal amplification step(s), IKK-mediated phosphorylation of the inhibitory IkB family proteins leads to its ubiquitination and proteasomal degradation, enabling the nuclear translocation and/or shuttling of NF-kB, eventually resulting in activation of target genes encoding proinflammatory cytokines (TNF-α, IL-1, and IL-6) and chemokines (IL-8).^[8] Interestingly, all TLRs, except TLR3, use the MyD88-dependent signaling pathway; on the contrary, the MyD88-independent pathway involves TRIF via the bridging adaptor TRIF-related adaptor molecule to activate NF-kB in either a TRAF-6-dependent fashion or a TRAF-6-independent manner, involving the kinase receptor-interacting protein (RIP)-1.^[7,8,9,12] Moreover, TRIF interacts with TRAF family member-associated NF-kB activator (TANK)-binding kinase 1 (TBK-1) and inducible IKB kinase (IKK-e, also known as IKK-i), which phosphorylate IRF-3 and IRF-7, leading to their nuclear translocation and subsequent induction of type 1 IFN genes.^[13] TRAF-3 is essential for the induction of type 1 IFN and the anti-inflammatory cytokine IL (IL)-10, and indispensable for expression of proinflammatory cytokines.^[7] TRAF-3 adaptor molecule, therefore, appears to be a critical “cellular/molecular signaling rheostat” for TRIF-mediated IRF-3 activation and IFN-beta production. Moreover, C5a, the C5 cleavage fragment of the complement system, has a negative influence on TLR4-induced IL-12, IL-23, and IL-27 syntheses from murine macrophages via extracellular signal regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)-dependent pathways.^[8] TLRs have been implicated in inflammatory disorders/diseases, viz., allergic airway inflammation, asthma, cancers (cervical, prostate, breast, gastric, and colorectal carcinomas), vasculoproliferative disorders including neointimal hyperplasia and restenosis, neurodegeneration, sepsis, and reproductive disorders.^[14] The immunotherapeutic role of TLRs in reproductive physiology and pathophysiology is now beginning to be recognized; therefore, future research studies are warranted using in vitro, in vivo, and patient-centric research approaches with a strong clinical impact so as to reduce the increasing prevalence of infertility in ethnic cohorts of diverse geographic locations and varying lifestyles.

TLR signaling in infertility

Unraveling the intriguing receptor-mediated cell signaling in oogenesis, spermatogenesis, fertilization, placentation, and implantation in human reproduction and disease is the objective of advanced research investigations/studies in biomedical sciences, primarily reproductive medicine field in both the United States of America, including States of Texas, New York, and Nebraska, and the Asian subcontinent, including North India. TLRs may prove to be a boon in the treatment of reproductive disorders; high throughput, sophisticated, cell and molecular biology-based studies will be immensely valuable in the eventual identification and validation of TLRs as patient-specific predictive and/or prognostic biomarkers.

TLRs, a family of evolutionarily conserved pathogen recognition receptors, are emerging as pivotal players in the pathophysiology of a spectrum of human diseases, including infertility. Inflammation and infections in the female reproductive tract are common causes of infertility globally.^[1] TLRs have significant roles in the immune system and initiate inflammatory response(s) to foreign pathogens including bacteria, viruses, and fungi, and are emerging as plausible susceptible markers in diverse human diseases, including reproductive disorders/diseases in both men and women.^[7] Public health research studies for developing cost-effective infertility assessment programs in low-resource settings targeting North Indian couples are ongoing so as to reduce the burden of disease as well as psychosocial factors associated with infertility.^[10] Significant recent advances in microarray and next-generation sequencing technologies have enabled the application of whole-genome approaches to the study of infertility. The physiology of human reproduction and disease is indeed complex, and scientific and clinical research efforts are warranted to provide a better understanding of the underlying cellular and molecular mechanisms involved in different phases of reproduction. Oogenesis involves a series of complex processes that produce a highly differentiated cell specialized for fertilization, with the distinct properties of a reduced chromosome complement, stored maternal mRNAs and energy precursors, pluripotency, and the ability to modify epigenetic marks on the paternal genome.^[11] A plethora of growth factors, many belonging to the TGF-beta superfamily, are expressed by ovarian somatic cells and oocytes in a stage-specific developmental manner and function as intraovarian regulators of folliculogenesis; bone morphogenetic proteins, BMP-4 and BMP-7, are expressed by ovarian stromal cells and/or theca cells and have recently been implicated as positive regulators of the primordial-to-primary follicle transition.^[15] Activin may play a positive role in oocyte maturation; in addition to its endocrine role to suppress Follicle Stimulating Hormone (FSH) secretion, increased output of inhibin by the selected dominant follicle(s) may upregulate Leutinizing Hormone (LH)-induced androgen secretion, which is required to sustain a relatively high level of estradiol secretion during the preovulatory phase.^[15] A majority of physiological events in spermatogenesis are well coordinated via signal amplification at the cell–cell interface through cell junctions, illustrating the significance of cell junctions and adhesion in spermatogenesis.^[16] Moreover, developing germ cells migrate across the seminiferous epithelium from the stem cell niche located in the basal compartment near the basement membrane of the tunica propria adjacent to the interstitium; additionally, synergistic and concerted actions of actin regulatory and polarity proteins may coordinate cyclic fluctuations of adhesion at Sertoli–Sertoli and Sertoli–germ cell interface(s) in the seminiferous epithelium during the epithelial cycle of spermatogenesis.^[16] Receptor-mediated cell signaling, including calcium-mediated signaling regulating inositol triphosphate-induced release from internal stores in biological systems, is a key aspect in oocytes’ response to fertilization in diverse species; initiation of embryo development depends on physiological [Ca²⁺]_i increase(s) in the egg, which is generally induced during fertilization.^[17] The physiological increase in concentration of intracellular calcium level(s) and spatiotemporal patterns signal egg activation, which is the first stage in embryo development, with subsequent structural and biochemical modifications that transform eggs into zygotes.^[17] Furthermore, the establishment of a successful pregnancy requires the implantation of a competent blastocyst into a “receptive” endometrium, facilitating the formation of a functional placenta.^[18] One of the major causes of infertility is inadequate implantation and placentation leading to first-trimester miscarriage(s), placental insufficiency, obstetric complications, etc.; embryo implantation comprises receptor-mediated and transmembrane cellular interactions between an implantation-competent blastocyst and a receptive uterus, which occurs in a limited time frame referred to as the window of implantation; aberrant physiological and/or metabolic defects originating during embryo implantation may lead to adverse consequences on later gestation events and unsuccessful pregnancy.^[19] Following fertilization (union of an egg with a sperm), the zygote undergoes multiple divisions and morphogenesis to form the next embryonic stage, blastocyst, with two distinct cell lineages: the outer specialized trophectodermal epithelium and the inner cell mass; the blastocyst participates in the first biophysical/physiological receptor-based and/or cell signaling-mediated interaction(s) with the maternal endometrium for initiation of implantation. Thereafter, subsequent bidirectional biochemical cross-talk(s) are essential for normal implantation and successful pregnancy, because signaling perturbations may generate adverse outcomes for further development, including decidualization and placentation, with potential loss of pregnancy. Early pregnancy loss occurring during the peri-implantation period before pregnancy is a relatively common biological phenomenon in humans.^[10]

Targeted manipulation of the TLR signaling pathway has immense practical/scientific utility; TLRs may be further validated as predictive biomarkers for diminished ovarian reserve, genital tuberculosis, and infertility in women of ethnically disparate populations, including North Indian women, thereby having wide economic implications in the future design of cost-effective public health infertility screening and disease management protocols in low-resource settings and accordingly stratifying susceptible individuals prior to in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) so as to have successful conception rates/pregnancy outcomes in women of developing countries. Inflammation and infections in the female reproductive tract are common causes of infertility globally; the activation of the innate immune system involving TLR2 and TLR2/6 in the uterus at the time of implantation has been demonstrated to interfere with the endometrial receptivity and thereby reduce the chances of implantation success.^[20] TLRs have been implicated in endometriosis/adverse reproductive outcomes.^[21] A recent study revealed that bacterial PAMPs initiate inflammation and perturb the endocrine function of bovine granulosa cells from dominant follicles via TLR2 and TLR4 pathways; supernatants of primary bovine granulosa cells accumulated IL-1β, IL-6, and IL-8 when treated for 24 h with Pam3CSK4 (PAM) that binds TLR2 or LPS that binds TLR4 but not flagellin that binds TLR5; granulosa cell responses to PAM or LPS were rapid, with increased phosphorylation of p38 and ERK1/2 within 30 min and increased abundance of IL6, IL1B, IL10, TNF, IL8, and CCL5 mRNA after 3 h of treatment, thereby strongly implicating TLR2 and TLR4 in endocrine-related disorders, including infertility.^[22] Furthermore, bacterial endotoxins, LPS, and peptidoglycan can be detected in human semen; the activated TLRs, primarily TLR2 and TLR4, reduce sperm motility, induce sperm apoptosis, and significantly impair the potential for fertilization.^[23] Although the cause of diminished ovarian reserve is multifactorial, it is possible that many cases attributed to an idiopathic cause may have a genetic component, including gene polymorphisms. A recent review on the genetic associations with diminished ovarian reserve implicated the emerging role of host genetic factors in infertility; one gene mutation (FMR1), three polymorphisms (GDF9, FSHR, and ESR1), and seven genes differentially expressed between women with diminished ovarian reserve and controls (AMH, LHCGR, IGF1, IGF2, IGF1R, IGF2R, and GREM1) were highlighted.^[24] TLR4 and MBL2 polymorphisms have been implicated in receptiveness to pathogens causing genital tract infections and susceptibility to tubal factor infertility; the TLR4 Asp299Gly and Thr399Ile heterozygosity was associated with a decreased incidence of pathogens associated with genital tract infections in tubal factor infertility patients.^[25] Furthermore, in hen ovarian follicles, theca cell layer expresses TLR2/4/5/7, while the granulosa cell layer expresses TLR4/55;^[26] TLR4 was detected in porcine ovary,^[27] and TLR5 is expressed primarily in human ovary, peripheral blood leukocytes, and prostate.^[28] Interestingly, laboratory data in murine models have demonstrated the expression of TLRs 2, 4, 8, and 9 in mouse granulosa cells and cumulus cells; TLR2 and TLR4 are functional because their ligands, Pam3Cys and LPS, respectively induce expression of known TLR2/TLR4 target genes, viz., Ptgs2, Il6, and TNF-α.^[29] Microarray database on cumulus–oocyte-complex (COC) samples collected from preovulatory follicles at 0 h, 8 h, and 16 h post-hCG revealed the presence of a majority of key components of the complex TLR signaling pathway; these include the genes encoding adaptor factors (CD14, Ly96, Myd88, Ticam1, Tirap, and Tollip), IRAKs, TNF-α receptor-associated factors (TRAFs), and IRFs, thereby strongly indicating the potential involvement of the TLR immune surveillance system in the ovulation process.^[28,29] Obesity may enhance the risk of developing infertility because of physiologically altered and/or aberrant TLR spatiotemporal function(s) in adipocytes that are associated with the release of immune cell-related factors, cytokines, and other metabolic products that regulate the components of the reproductive system, including ovary.^[30] Genetic studies are essential to identify/stratify disease-susceptible individuals in a population; host genetic differences that influence the primary immune response may determine those who are at increased risk for progression to clinical infertility. It is now becoming increasingly apparent that TLRs have considerable role in reproductive/endocrine diseases as well as gynecologic malignancies, including Human Papillomavirus-mediated cervical cancer.^[31] In this context, association analyses in a population setting can be used to explore the role of genetic polymorphisms in susceptibility to various reproductive/endocrine disorders, including clinical infertility. Moreover, early identification of infertility-susceptible individuals at the population level may be successfully achieved by TLR-based therapeutics and TLR-based biomarker studies in an ethnically disparate cohort.^[31]

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Conflicts of interest

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