Tatyana O. Volkova

Mannose receptor 1 mediates cellular uptake and endosomal delivery of CpG-motif containing oligodeoxynucleotides.

Recognition of microbial components is critical for activation of TLRs, subsequent innate immune signaling, and directing adaptive immune responses. The DNA sensor TLR9 traffics from the endoplasmic reticulum to endolysosomal compartments where it is cleaved by resident proteases to generate a competent receptor. Activation of TLR9 by CpG-motif containing oligodeoxynucleotides (CpG ODNs) is preceded by agonist endocytosis and delivery into the endolysosomes. The events that dictate this process remain largely unknown; furthermore, it is unclear whether the receptors involved in mediating uptake of exogenous DNA are conserved for both naturally derived pathogenic DNA and synthetic ODNs. In this study, we report that peritoneal macrophages from a wild-derived inbred mouse strain, MOLF/Ei, are hyporesponsive to CpG ODN but are fully responsive to bacterial DNA, thus implying that microbial recognition is not fully recapitulated by a synthetic analog. To identify the gene responsible for the CpG ODN defect, we have performed genome-wide linkage analysis. Using N2 backcross mice, we mapped the trait with high resolution to a single locus containing Mrc1 as the gene conferring the trait. We show that mannose receptor 1 (MRC1; CD206) is involved in CpG ODN uptake and trafficking in wild-derived MOLF/Ei peritoneal macrophages. Furthermore, we show that other strains of wild-derived mice also require MRC1 for CpG-induced cytokine responses. These findings reveal novel functions for MRC1 and demonstrate that wild-derived mice are important and indispensable model for understanding naturally occurring regulators of inflammatory responses in innate immune pathways.

Balance between short and long isoforms of cFLIP regulates Fas-mediated apoptosis in vivo

Significance To our knowledge, this article is the first report explaining how cFLIP, an inhibitor of apoptosis, regulates apoptosis in vivo. Although the antiapoptotic role of cFLIP was proposed based on in vitro studies and the early embryonic lethality of cFLIP-deficient mice, the specific role of cFLIPL (long) and cFLIPR (short) isoforms is poorly understood. In this study, we describe a previously unidentified allele of caspase 8- and FADD-like apoptosis regulator (Cflar) (encoding cFLIP) that makes mice of MSM strain resistant to Fas-mediated lethality. The mutant allele affects the ratio of cFLIPL:cFLIPR, leading to high levels of long FLIP in MSM. As a result, the abundant cFLIPL forms enzymatically active heterodimers with caspase 8 (CASP8) in MSMs, which prevents formation of proapoptotic CASP8 p10/p20 and cleaves receptor interacting protein kinase 1 (RIP1), thus setting up a higher threshold for CD95-mediated apoptosis and RIP1-mediated necroptosis. cFLIP, an inhibitor of apoptosis, is a crucial regulator of cellular death by apoptosis and necroptosis; its importance in development is exemplified by the embryonic lethality in cFLIP–deficient animals. A homolog of caspase 8 (CASP8), cFLIP exists in two main isoforms: cFLIPL (long) and cFLIPR (short). Although both splice variants regulate death receptor (DR)-induced apoptosis by CASP8, the specific role of each isoform is poorly understood. Here, we report a previously unidentified model of resistance to Fas receptor-mediated liver failure in the wild-derived MSM strain, compared with susceptibility in C57BL/6 (B6) mice. Linkage analysis in F2 intercross (B6 x MSM) progeny identified several MSM loci controlling resistance to Fas-mediated death, including the caspase 8- and FADD-like apoptosis regulator (Cflar) locus encoding cFLIP. Furthermore, we identified a 21-bp insertion in the 3′ UTR of the fifth exon of Cflar in MSM that influences differential splicing of cFLIP mRNA. Intriguingly, we observed that MSM liver cells predominantly express the FLIPL variant, in contrast to B6 liver cells, which have higher levels of cFLIPR. In keeping with this finding, genome-wide RNA sequencing revealed a relative abundance of FLIPL transcripts in MSM hepatocytes whereas B6 liver cells had significantly more FLIPR mRNA. Importantly, we show that, in the MSM liver, CASP8 is present exclusively as its cleaved p43 product, bound to cFLIPL. Because of partial enzymatic activity of the heterodimer, it might prevent necroptosis. On the other hand, it prevents cleavage of CASP8 to p10/20 necessary for cleavage of caspase 3 and, thus, apoptosis induction. Therefore, MSM hepatocytes are predisposed for protection from DR-mediated cell death.

Cutting Edge: Novel Tmem173 Allele Reveals Importance of STING N Terminus in Trafficking and Type I IFN Production.

With the stimulator of IFN genes (STING) C terminus being extensively studied, the role of the N-terminal domain (NTD) of STING remains an important subject of investigation. In this article, we identify novel mutations in NTD of Sting of the MOLF strain in response to HSV and Listeria monocytogenes both in vitro and in vivo. These mutations are responsible for low levels of IFN-β caused by failure of MOLF STING to translocate from the endoplasmic reticulum. These data provide evidence that the NTD of STING affects DNA responses via control of trafficking. They also show that the genetic diversity of wild-derived mice resembles the diversity observed in humans. Several human alleles of STING confer attenuated IFN-I production similar to what we observe with the MOLF Sting allele, a crucial functional difference not apparent in classical inbred mice. Thus, understanding the functional significance of polymorphisms in MOLF STING can provide basic mechanistic insights relevant to humans.

T- and NK-cell populations with regulatory phenotype and markers of apoptosis in circulating lymphocytes of patients with CIN3 or microcarcinoma of the cervix: evidence for potential mechanisms of immune suppression

BackgroundProcesses and mechanisms responsible for systemic immune suppression in early-stage cervical cancer remain substantially underinvestigated. In this work, we focused on studying the frequencies of circulating regulatory T (CD4 and CD8 Tregs) and NK (NKregs) cells in parallel with assessment of apoptotic markers expression in T cells from patients with preinvasive and microinvasive cervical cancer, with the aim to determine whether up-regulation of apoptosis-associated markers in Т lymphocytes accompanies cervical cancer development and correlates with the change in percentages of regulatory cell populations at systemic level during the initial stages of invasive cervical cancer progression.MethodsFourty two women with histologically confirmed cervical intraepithelial neoplasia grade 3 (CIN3, including carcinoma in situ) or cervical cancer (stage IA) and 30 healthy women (control) were enrolled in the study. Peripheral blood samples were taken immediately before surgery or any treatment and immediately subjected to multicolor flow cytometry.ResultsAnalysis of a combination of CD4/CD8, CD25, CD127, and FoxP3 markers revealed a statistically significant increase in the frequencies of Tregs within both the CD4 and CD8 subsets of circulating lymphocytes in patients with CIN3 and stage IA cancer. In contrast, lower numbers of NKregs (defined as CD16dim/negCD56bright subpopulation) and increased CD56dim/CD56bright NK ratio were found in patients compared to controls, with the percentage of CD16brightCD56dim cells (major subtype of circulating NKs) showing no difference. Patients also exhibited an increased expression of CD95 in total peripheral blood T lymphocytes, along with increased level of Annexin V binding to CD95-positive cells, suggesting higher susceptibility of T cells to apoptosis and potential involvement of CD95-dependent pathway in early-stage cervical cancer. Differential analysis of CD4 and CD8 T cells revealed different trends in the change of CD95 expression, confirming that this change likely has different functional significance for these two subsets. A search for correlations between the phenotypic parameters analyzed in this study was performed to demonstrate that women with early neoplastic lesions of the cervix, such as carcinoma in situ and microinvasive carcinoma, displayed a coordinated increase in expression of Treg markers in circulating lymphocytes, along with more pronounced cross-relationships between Treg numbers, CD95 expression on T cells, and apoptosis, compared to the control group.ConclusionsThe results of this study suggest that a diversity of immune regulatory mechanisms that provide support for initial stages of invasive growth in cervical cancer patients includes systemic changes in the ratios between the principal regulatory and effector lymphocyte populations both within adaptive and innate immunity.

Caspases as Putative Biomarkers of Cervical Cancer Development

Resistance to apoptosis is commonly accepted as the principal hallmark of a cancer cell, while caspases are recognized as the key molecular players of the apoptosis regulatory network. Since the level of caspase activity is thought to be directly coupled with aggres‐ sive features of cancer cells (such as ability to withstand immune reactions, invasiveness, drug resistance, etc.), these proteases could serve as objective diagnostic markers espe‐ cially for those types of cancer where early differential diagnosis is needed. Cervical can‐ cer develops through morphologically well-described stages—from intraepithelial lesions of 1/2/3 grade including carcinoma in situ to microinvasive and invasive cancer with pre‐ cancerous lesions known to be potentially reversible. The percentage of cervical neo‐ plasms diagnosed at early stages is relatively high, providing a basis for the use of cervical cancer as an in vivo model to investigate the mechanisms of apoptosis modula‐ tion in malignant cells. The existing diagnostic criteria, despite their usefulness, have sub‐ stantial limitations with respect to cervical cancer and preneoplastic lesions, so caspases may be helpful in improving them, but there is insufficient data regarding the involve‐ ment of these enzymes in cervical cancer development. In this chapter, we report on spe‐ cific patterns of activity of caspases revealed in tissue biopsies and blood lymphocytes in association with different stages of cervical cancer development. The data indicate that caspases are pivotal components of the in vivo molecular “portrait” of cervical cancer and have the potential of being used as biomarkers.

EXPERIMENTAL APPROACHES TO DERIVING PRIMARY TUMOR CELL CULTURES FROM CERVICAL CANCER BIOPSIES: A COMPARATIVE ANALYSIS OF PUBLISHED DATA AND OWN EXPERIENCE

The article provides a detailed description of the procedure of converting primary tumor cells of invasive cervical cancer into a state of a transient in vitro culture, including the description of conditions for tissue dissociation and selection of appropriate cell culture medium. Morphological characteristics of the cell colonies obtained and the results of their phenotyping (based on analysis of cytokeratin expression) are presented. Our own observations are discussed in comparison with the previously published studies aimed at defining optimal conditions to culture primary cells of cervical neoplasia.

Cellular Caspases: New Targets for the Action of Pharmacological Agents

The number of scientific papers devoted to the study of caspases has lately being constantly growing. Caspases are a family of cysteine-dependent aspartate specific proteases. Caspases play an essential role in the apoptosis, necrosis and inflammation processes. Apoptosis is asynchronous programmed cell death, which helps maintain the physiological balance and genetic stability of the organism through self-destruction of genetically modified defect cells. Apoptosis may happen also in normal cells, e.g., during embryogenesis. During apoptosis, activated endogenous nucleases cleave DNA into fragments, but cell membranes and the intracellular matter remain intact, nor is there tissue damage or leukocytic infiltration. In contrast to apoptosis, necrosis is a pathological form of cell death caused by acute damage, rupture of the membrane, release of the cytoplasm content, and the inflammatory process induced thereby [1, 2].