Correction: Hu et al. Profiles of Long Non-Coding RNAs and mRNA Expression in Human Macrophages Regulated by Interleukin-27. Int. J. Mol. Sci. 2019, 20, 6207.

The authors wish to make the following corrections to this paper [...].

MicroRNA Profiles in Monocyte-Derived Macrophages Generated by Interleukin-27 and Human Serum: Identification of a Novel HIV-Inhibiting and Autophagy-Inducing MicroRNA.

Interleukin-27 (IL-27) is a pleiotropic cytokine that influences the innate and adaptive immune systems. It inhibits viral infection and regulates the expression of microRNAs (miRNAs). We recently reported that macrophages differentiated from human primary monocytes in the presence of IL-27 and human AB serum resisted human immunodeficiency virus (HIV) infection and showed significant autophagy induction. In the current study, the miRNA profiles in these cells were investigated, especially focusing on the identification of novel miRNAs regulated by IL-27-treatment. The miRNA sequencing analysis detected 38 novel miRNAs. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed that IL-27 differentially regulated the expression of 16 of the 38 miRNAs. Overexpression of the synthesized miRNA mimics by transfection revealed that miRAB40 had potent HIV-inhibiting and autophagy-inducing properties. B18R, an interferon (IFN)-neutralization protein, partially suppressed both activities, indicating that the two functions were induced via IFN-dependent and -independent pathways. Although the target mRNA(s) of miRAB40 involving in the induction of both functions was unable to identify in this study, the discovery of miRAB40, a potential HIV-inhibiting and autophagy inducing miRNA, may provide novel insights into the miRNA (small none-coding RNA)-mediated regulation of HIV inhibition and autophagy induction as an innate immune response.

Regulators of the protein phosphatase PP1γ2, PPP1R2, PPP1R7, and PPP1R11 are involved in epididymal sperm maturation.

The serine/threonine protein phosphatase 1 (PP1) inhibitors PPP1R2, PPP1R7, and PPP1R11 are evolutionarily ancient and highly conserved proteins. Four PP1 isoforms, PP1α, PP1ß, PP1γ1, and PP1γ2, exist; three of them except PP1γ2 are ubiquitous. The fact that PP1γ2 isoform is present only in mammalian testis and sperm led to the notion that isoform-specific regulators for PP1γ2 in sperm may be responsible for its function. In this report, we studied these inhibitors, PPP1R2, R7, and R11, to determine their spatial and temporal expression in testis and their regulatory functions in sperm. We show that, similar to PP1γ2, the three inhibitors are expressed at high levels in developing spermatogenic cells. However, the transcripts for the regulators are expressed as unique sizes in testis compared with somatic tissues. The three regulators share localization with PP1γ2 in the head and the principal piece of sperm. We show that the association of inhibitors to PP1γ2 changes during epididymal sperm maturation. In immotile caput epididymal sperm, PPP1R2 and PPP1R7 are not bound to PP1γ2, whereas in motile caudal sperm, all three inhibitors are bound as heterodimers or heterotrimers. In caudal sperm from male mice lacking sAC and glycogen synthase kinase 3, where motility and fertility are impaired, the association of PP1γ2 to the inhibitors resembles immature caput sperm. Changes in the association of the regulators with PP1γ2, due to their phosphorylation, are part of biochemical mechanisms responsible for the development of motility and fertilizing ability of sperm during their passage through the epididymis.

The protein phosphatase isoform PP1γ1 substitutes for PP1γ2 to support spermatogenesis but not normal sperm function and fertility†.

Four isoforms of serine/threonine phosphatase type I, PP1α, PP1ß, PP1γ1, and PP1γ2, are derived from three genes. The PP1γ1 and PP1γ2 isoforms are alternately spliced transcripts of the protein phosphatase 1 catalytic subunit gamma gene (Ppp1cc). While PP1γ1 is ubiquitous in somatic cells, PP1γ2 is expressed exclusively in testicular germ cells and sperm. Ppp1cc knockout male mice (-/-), lacking both PP1γ1 and PP1γ2, are sterile due to impaired sperm morphogenesis. Fertility and normal sperm function can be restored by transgenic expression of PP1γ2 alone in testis of Ppp1cc (-/-) mice. The purpose of this study was to determine whether the PP1γ1 isoform is functionally equivalent to PP1γ2 in supporting spermatogenesis and male fertility. Significant levels of transgenic PP1γ1 expression occurred only when the transgene lacked a 1-kb 3΄UTR region immediately following the stop codon of the PP1γ1 transcript. PP1γ1 was also incorporated into sperm at levels comparable to PP1γ2 in sperm from wild-type mice. Spermatogenesis was restored in mice expressing PP1γ1 in the absence of PP1γ2. However, males from the transgenic rescue lines were subfertile. Sperm from the PP1γ1 rescue mice were unable to fertilize eggs in vitro. Intrasperm localization of PP1γ1 and the association of the protein regulators of the phosphatase were altered in epididymal sperm in transgenic PP1γ1 compared to PP1γ2. Thus, the ubiquitous isoform PP1γ1, not normally expressed in differentiating germ cells, could replace PP1γ2 to support spermatogenesis and spermiation. However, PP1γ2, which is the PP1 isoform in mammalian sperm, has an isoform-specific role in supporting normal sperm function and fertility.

Profiles of Long Non-Coding RNAs and mRNA Expression in Human Macrophages Regulated by Interleukin-27.

Macrophages play an essential role in the immune system. Recent studies have shown that long non-coding RNAs (lncRNAs) can regulate genes encoding products involved in the immune response. Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines with broad anti-viral effects that inhibits human immunodeficiency virus (HIV) type-1 and herpes simplex virus (HSV). However, little is known about the role of lncRNAs in macrophages affected by IL-27. Therefore, we investigated the expression profiles of mRNA and lncRNA in human monocyte-derived macrophages (MDMs) regulated by IL-27. Monocytes were differentiated in the presence of macrophage-colony stimulatory factor (M-CSF)- or human AB serum with or without IL-27, and these cells were the subject for the profile analysis using RNA-Seq. We identified 146 lncRNAs (including 88 novel ones) and 434 coding genes were differentially regulated by IL-27 in both M-CSF- and AB serum-induced macrophages. Using weighted gene co-expression network analysis, we obtained four modules. The immune system, cell cycle, and regulation of complement cascade pathways were enriched in different modules. The network of mRNAs and lncRNAs in the pathways suggest that lncRNAs might regulate immune activity in macrophages. This study provides potential insight into the roles of lncRNA in macrophages regulated by IL-27.

Cyclic AMP and glycogen synthase kinase 3 form a regulatory loop in spermatozoa.

The multifaceted glycogen synthase kinase (GSK3) has an essential role in sperm and male fertility. Since cyclic AMP (cAMP) plays an important role in sperm function, we investigated whether GSK3 and cAMP pathways may be interrelated. We used GSK3 and soluble adenylyl cyclase (sAC) knockout mice and pharmacological modulators to examine this relationship. Intracellular cAMP levels were found to be significantly lower in sperm lacking GSK3α or GSK3ß. A similar outcome was observed when sperm cells were treated with SB216763, a GSK3 inhibitor. This reduction of cAMP levels was not due to an effect on sperm adenylyl cyclase but was caused by elevated phosphodiesterase (PDE) activity. The PDE4 inhibitor RS25344 or the general PDE inhibitor IBMX could restore cAMP levels in sperm lacking GSK3α or ß-isoform. PDE activity assay also showed that hyperactivated PDE4 contributes in lowering of cAMP levels in GSK3α null sperm suggesting that in wild-type sperm PDE4 activity is kept in check by GSK3. Conversely, PKA being triggered by cAMP, affected GSK3 activity through increasing its phosphorylation. Increased GSK3 phosphorylation also occurred by inhibition of sperm specific protein phosphatase type 1, PP1γ2. The relationship between cAMP, GSK3, and PP1γ2 activities was also confirmed in sperm from sAC null mice. Pull-down assay using recombinant PP1γ2 indicated that PKA, GSK3, and PP1γ2 could exist as a complex. Pharmacological inhibition of GSK3 in mature spermatozoa resulted in significantly reduced fertilization of eggs in vitro. Our results show that cAMP, PKA, and GSK3 are interrelated in regulation of sperm function.

Isoform-specific requirement for GSK3α in sperm for male fertility.

Glycogen synthase kinase 3 (GSK3) is a highly conserved protein kinase regulating key cellular functions. Its two isoforms, GSK3α and GSK3ß, are encoded by distinct genes. In most tissues the two isoforms are functionally interchangeable, except in the developing embryo where GSK3ß is essential. One functional allele of either of the two isoforms is sufficient to maintain normal tissue functions. Both GSK3 isoforms, present in sperm from several species including human, are suggested to play a role in epididymal initiation of sperm motility. Using genetic approaches, we have tested requirement for each of the two GSK3 isoforms in testis and sperm. Both GSK3 isoforms are expressed at high levels during the onset of spermatogenesis. Conditional knockout of GSK3α, but not GSK3ß, in developing testicular germ cells in mice results in male infertility. Mice lacking one allele each of GSK3α and GSK3ß are fertile. Despite overlapping expression and localization in differentiating spermatids, GSK3ß does not substitute for GSK3α. Loss of GSK3α impairs sperm hexokinase activity resulting in low ATP levels. Net adenine nucleotide levels in caudal sperm lacking GSK3α resemble immature caput epididymal sperm. Changes in the association of the protein phosphatase PP1γ2 with its protein interactors occurring during epididymal sperm maturation is impaired in sperm lacking GSK3α. The isoform-specific requirement for GSK3α is likely due to its specific binding partners in the sperm principal piece. Testis and sperm are unique in their specific requirement of GSK3α for normal function and male fertility.

Targeted disruption of glycogen synthase kinase 3A (GSK3A) in mice affects sperm motility resulting in male infertility.

The signaling enzyme glycogen synthase kinase 3 (GSK3) exists as two isoforms-GSK3A and GSK3B. Protein phosphorylation by GSK3 has important signaling roles in several cells. In our past work, we found that both isoforms of GSK3 are present in mouse sperm and that catalytic GSK3 activity correlates with motility of sperm from several species. Here, we examined the role of Gsk3a in male fertility using a targeted gene knockout (KO) approach. The mutant mice are viable, but have a male infertility phenotype, while female fertility is unaffected. Testis weights of Gsk3a(-/-) mice are normal and sperm are produced in normal numbers. Although spermatogenesis is apparently unimpaired, sperm motility parameters in vitro are impaired. In addition, the flagellar waveform appears abnormal, characterized by low amplitude of flagellar beat. Sperm ATP levels were lower in Gsk3a(-/-) mice compared to wild-type animals. Protein phosphatase PP1 gamma2 protein levels were unaltered, but its catalytic activity was elevated in KO sperm. Remarkably, tyrosine phosphorylation of hexokinase and capacitation-associated changes in tyrosine phosphorylation of proteins are absent or significantly lower in Gsk3a(-/-) sperm. The GSK3B isoform was present and unaltered in testis and sperm of Gsk3a(-/-) mice, showing the inability of GSK3B to substitute for GSK3A in this context. Our studies show that sperm GSK3A is essential for male fertility. In addition, the GSK3A isoform, with its highly conserved glycine-rich N terminus in mammals, may have an isoform-specific role in its requirement for normal sperm motility and fertility.

Short Communication: S100A8 and S100A9, Biomarkers of SARS-CoV-2 Infection and Other Diseases, Suppress HIV Replication in Primary Macrophages.

S100A8 and S100A9 are members of the Alarmin family; these proteins are abundantly expressed in neutrophils, form a heterodimer complex, and are secreted in plasma on pathogen infection or acute inflammatory diseases. Recently, both proteins were identified as novel biomarkers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and were shown to play key roles in inducing an aggressive inflammatory response by mediating the release of large amounts of pro-inflammatory cytokines, called the "cytokine storm." Although co-infection with SARS-CoV-2 in people living with HIV-1 may result in an immunocompromised status, the role of the S100A8/A9 complex in HIV-1 replication in primary T cells and macrophages is still unclear. Here, we evaluated the roles of the proteins in HIV replication to elucidate their functions. We found that the complex had no impact on virus replication in both cell types; however, the subunits of S100A8 and S100A9 inhibit HIV in macrophages. These findings provide important insights into the regulation of HIV viral loads during SARS-CoV-2 co-infection.

Complete Genome Sequence of Herpes Simplex Virus 2 Strain G.

Herpes simplex virus type 2 (HSV-2) is a common causative agent of genital tract infections. Moreover, HSV-2 and HIV infection can mutually increase the risk of acquiring another virus infection. Due to the high GC content and highly repetitive regions in HSV-2 genomes, only the genomes of four strains have been completely sequenced (HG52, 333, SD90e, and MS). Strain G is commonly used for HSV-2 research, but only a partial genome sequence has been assembled with Illumina sequencing reads. In the current study, we de novo assembled and annotated the complete genome of strain G using PacBio long sequencing reads, which can span the repetitive regions, analyzed the 'α' sequence, which plays key roles in HSV-2 genome circulation, replication, cleavage, and packaging of progeny viral DNA, identified the packaging signals homologous to HSV-1 within the 'α' sequence, and determined both termini of the linear genome and cleavage site for the process of concatemeric HSV-2 DNA produced via rolling-circle replication. In addition, using Oxford Nanopore Technology sequencing reads, we visualized four HSV-2 genome isomers at the nucleotide level for the first time. Furthermore, the coding sequences of HSV-2 strain G have been compared with those of HG52, 333, and MS. Moreover, phylogenetic analysis of strain G and other diverse HSV-2 strains has been conducted to determine their evolutionary relationship. The results will aid clinical research and treatment development of HSV-2.

S100A8 and S100A9, biomarkers of SARS-Cov2-infected patients, suppress HIV replication in primary macrophages.

S100A8 and S100A9 are members of the Alarmin family; these proteins are abundantly expressed in neutrophils and form a heterodimer complex. Recently, both proteins were identified as novel biomarkers of SARS-CoV-2 infection and were shown to play key roles in inducing an aggressive inflammatory response by mediating the release of large amounts of pro-inflammatory cytokines, called the "cytokine storm." Although co-infection with SARS-CoV-2 in people living with HIV-1 may result in an immunocompromised status, the role of the S100A8/A9 complex in HIV-1 replication in primary T cells and macrophages is still unclear. Here, we evaluated the roles of the proteins in HIV replication to elucidate their functions. We found that the complex had no impact on virus replication in both cell types; however, the subunits of S100A8 and S100A9 inhibits HIV in macrophages. These findings provide important insights into the regulation of HIV viral loads in SARS-CoV2 co-infection.

Profiles of MicroRNAs in Interleukin-27-Induced HIV-Resistant T Cells: Identification of a Novel Antiviral MicroRNA.

OBJECTIVES: Interleukin-27 (IL-27) is known as an anti-HIV cytokine. We have recently demonstrated that IL-27-pretreatment promotes phytohemagglutinin-stimulated CD4(+) T cells into HIV-1-resistant cells by inhibiting an uncoating step. PURPOSE: To further characterize the function of the HIV resistant T cells, we investigated profiles of microRNA in the cells using microRNA sequencing (miRNA-seq) and assessed anti-HIV effect of the microRNAs. METHODS: Phytohemagglutinin-stimulated CD4(+) T cells were treated with or without IL-27 for 3 days. MicroRNA profiles were analyzed using miRNA-seq. To assess anti-HIV effect, T cells or macrophages were transfected with synthesized microRNA mimics and then infected with HIVNL4.3 or HIVAD8. Anti-HIV effect was monitored by a p24 antigen enzyme-linked immunosorbent assay kit. interferon (IFN)-α, IFN-ß, or IFN-λ production was quantified using each subtype-specific enzyme-linked immunosorbent assay kit. RESULTS: A comparative analysis of microRNA profiles indicated that expression of known miRNAs was not significantly changed in IL-27-treated cells compared with untreated T cells; however, a total of 15 novel microRNAs (miRTC1 ∼ miRTC15) were identified. Anti-HIV assay using overexpression of each novel microRNA revealed that 10 nM miRTC14 (GenBank accession number: MF281439) remarkably suppressed HIV infection by (99.3 ± 0.27%, n = 9) in macrophages but not in T cells. The inhibition was associated through induction of >1000 pg/mL of IFN-αs and IFN-λ1. CONCLUSION: We discovered a total of 15 novel microRNAs in T cells and characterized that miRTC14, one of the novel microRNAs, was a potent IFN-inducing anti-HIV miRNA, implicating that regulation of the expression of miRTC14 may be a potent therapeutic tool for not only HIV but also other virus infection.

A Combination of M50I and V151I Polymorphic Mutations in HIV-1 Subtype B Integrase Results in Defects in Autoprocessing.

We have recently reported that a recombinant HIV-1NL4.3 containing Met-to-Ile change at codon 50 of integrase (IN) (IN:M50I) exhibits suppression of the virus release below 0.5% of WT HIV, and the released viral particles are replication-incompetent due to defects in Gag/GagPol processing by inhibition of the initiation of autoprocessing of GagPol polyproteins in the virions and leads to replication-incompetent viruses. The coexisting Ser-to-Asn change at codon 17 of IN or Asn-to-Ser mutation at codon 79 of RNaseH (RH) compensated the defective IN:M50I phenotype, suggesting that both IN and RH regulate an HIV infectability. In the current study, to elucidate a distribution of the three mutations during anti-retroviral therapy among patients, we performed a population analysis using 529 plasma virus RNA sequences obtained through the MiSeq. The result demonstrated that 14 plasma HIVs contained IN:M50I without the compensatory mutations. Comparing the sequences of the 14 viruses with that of the defective virus illustrated that only Val-to-Ile change at codon 151 of IN (IN:V151I) existed in the recombinant virus. This IN:V151I is known as a polymorphic mutation and was derived from HIVNL4.3 backbone. A back-mutation at 151 from Ile-to-Val in the defective virus recovered HIV replication capability, and Western Blotting assay displayed that the back-mutation restored Gag/GagPol processing in viral particles. These results demonstrate that a combination of IN:M50I and IN:V151I mutations, but not IN:M50I alone, produces a defective virus.

IL-27 posttranslationally regulates Y-box binding protein-1 to inhibit HIV-1 replication in human CD4+ T cells.

OBJECTIVES: IL-27 is known as an antiviral cytokine that inhibits HIV, hepatitis C virus, and other viruses. We have previously demonstrated that, IL-27 posttreatment after HIV-infection inhibits viral replication in primary CD4 T cells. DESIGN: Here, we evaluated the anti-HIV effect of IL-27 pretreatment in CD4 T cells from healthy donors prior to HIV infection with HIVNL4.3 or vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-luciferase virus (HIV-LUC-V). METHODS: IL-27-treated CD4 T cells were infected with HIVNL4.3 or HIV-LUC-V and assessed the anti-HIV effect. HIV infection was monitored by p24 antigen ELISA or luciferase assay. HIV fusion/entry and uncoating were determined by BlaM-Vpr assay and HIV fate of capsid and/or HIV Entry/Uncoating assay based on core-packaged RNA availability and Translation assay, respectively. HIV proviral copy number was determined by real-time PCR. Gene expression profile from IL-27-pretreated CD4 T cells was determined using Genechip array. Posttranslational modification of global proteins from IL-27-pretreated CD4 T cells was determined by a combination of 2-dimensional difference-in-gel-electrophoresis (2D-DIG), western-blot and protein mass spectrometry. RESULTS: IL-27 pretreatment inhibited HIVNL4.3 and HIV-LUC-V infection in CD4 T cells. HIV copy assay demonstrated that IL-27-treatment suppressed an early step of reverse transcription during HIV infection. A combination of 2D-DIG-electrophoresis and western blot assays demonstrated that IL-27-treatment induces a change in posttranslational modification of Y box binding protein-1 (YB-1). Overexpression of domain negative YB-1 mutants illustrated that a residue Lysine at 118 plays a key role in supporting HIV infection in CD4 T cells. CONCLUSION: IL-27-pretreatment inhibits HIV-1 infection by suppressing an HIV-reverse transcription product formation/uncoating step by suppressing the acetylation of YB-1 in primary CD4 T cells.

Copper induced immunotoxicity promote differential apoptotic pathways in spleen and thymus.

Inorganic copper, such as that in drinking water and copper supplements, largely bypasses the liver and enters the free copper pool of the blood directly and that promote immunosuppression. Nevertheless, the signaling pathways underlying copper-induced immune cell death remains largely unclear. According to our previous in vivo report, to evaluate the further details of the apoptotic mechanism, we have investigated how copper regulates apoptotic pathways in spleen and thymus. We have analyzed different protein expression by western blotting and immunohistochemistry and mRNA expression by RT-PCR and gel electrophoresis. We also have measured mitochondrial trans-membrane potential, ROS and CD4(+) and CD8(+) population by flow cytometry. Sub lethal doses of copper in spleen and thymus of in vivo Swiss albino mice promote different apoptotic pathways. In case of spleen, ROS generation and mitochondrial trans-membrane potential changes promotes intrinsic pathway of apoptosis that was p53 independent, ultimately leads to decrease in CD4(+) T cell population and increase in CD8(+) T cell population. However in case of thymus, ROS generation and mitochondrial trans-membrane potential changes lead to death receptor that regulate extrinsic and intrinsic pathways of apoptosis and the apoptotic mechanism which was p53 dependent. Due to copper treatment, thymic CD4(+) T cell population decreased and CD8(+) T cell population was increased or proliferated. Apart from the role of inflammation, our findings also have identified the role of other partially responsible apoptotic molecules like p27, p73, p62, poly (ADP-ribose) polymerase (PARP) that differentially changed due to copper treatment in spleen and thymus of Swiss albino mice. Present study firstly demonstrates how apoptotic pathways differentially regulate copper induced immunosuppression.

Copper-induced immunotoxicity involves cell cycle arrest and cell death in the spleen and thymus.

Copper is an essential trace element for human physiological processes. To evaluate the potential adverse health impact/immunotoxicological effects of this metal in situ due to over exposure, Swiss albino mice were treated (via intraperitoneal injections) with copper (II) chloride (copper chloride) at doses of 0, 5, or 7.5 mg copper chloride/kg body weight (b.w.) twice a week for 4 wk; these values were derived from LD50 studies using copper chloride doses that ranged from 0 to 40 mg/kg BW (2×/wk, for 4 wk). Copper treated mice evidenced immunotoxicity as indicated by dose-related decreases and increases, respectively, in thymic and splenic weights. Histomorphological changes evidenced in these organs were thymic atrophy, white pulp shrinkage in the spleen, and apoptosis of splenocytes and thymocytes; these observations were confirmed by microscopic analyses. Cell count analyses indicated that the proliferative functions of the splenocytes and thymocytes were also altered because of the copper exposures. Among both cell types from the copper treated hosts, flow cytometric analyses revealed a dose related increase in the percentages of cells in the Sub-G0/G1 state, indicative of apoptosis which was further confirmed by Annexin V binding assay. In addition, the copper treatments altered the expression of selected cell death related genes such as EndoG and Bax in a dose related manner. Immunohistochemical analyses revealed that there was also increased ubiquitin expression in both the cell types. In conclusion, these studies show that sublethal exposure to copper (as copper chloride) induces toxicity in the thymus and spleen, and increased Sub G0/G1 population among splenocytes and thymocytes that is mediated, in part, by the EndoG-Bax-ubiquitin pathway. This latter damage to these cells that reside in critical immune system organs are likely to be important contributing factors underlying the immunosuppression that has been documented by other investigators following acute high dose/chronic low-medium dose exposures to copper agents.