Recognition of microbial components is critical for activation of Toll-like receptors (TLRs), subsequent innate immune signaling and directing adaptive immune responses. The DNA sensor TLR9 traffics from the endoplasmic reticulum (ER) to endolysosomal compartments where it is cleaved by resident proteases to generate a competent receptor. Activation of TLR9 by CpG ODN 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. Here we report that peritoneal macrophages from a wild-derived inbred mouse strain, MOLF/Ei, are hypo-responsive 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 MRC1 (mannose receptor, 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.

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: cFLIP L (long) and cFLIP R (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 FLIP L variant, in contrast to B6 liver cells, which have higher levels of cFLIP R. In keeping with this finding, genome-wide RNA sequencing revealed a relative abundance of FLIP L transcripts in MSM hepatocytes whereas B6 liver cells had significantly more FLIP R mRNA. Importantly, we show that, in the MSM liver, CASP8 is present exclusively as its cleaved p43 product, bound to cFLIP L . 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.T he Fas receptor [also called cluster of differentiation 95 (CD95), APO-1, or TNFRSF6] is a death receptor family member constitutively expressed by most tissues, including the liver (1), where ligation of ubiquitously expressed CD95 leads to potentially lethal hepatitis and liver failure. Although CD95L (Fas ligand) is the only known physiological ligand of CD95 (2), the agonistic antibody Jo2 has been used extensively to ligate CD95 and model CD95-mediated hepatotoxicity and mortality in mice (3). In contrast to the ability of tumor necrosis factor receptor (TNFR)-mediated signaling to lead to profound inflammatory responses in addition to cell death (4), CD95 is predominantly used in apoptosis and necrosis and therefore engages a limited number of downstream components (5). Specifically, ligand binding induces CD95 oligomerization and binding of Fas-associated death domain (FADD) via its death domain (DD), which then recruits caspase 8 (CASP8) via a death effector domain (DED), forming the death-inducing signaling complex (DISK) comprising receptor interacting protein kinase 1 (RIP1), FADD, and CASP8 (6).Interactions of caspase 8 (CASP8) with its enzymatically inactive homolog cFLIP L further complicate the regulation of CD95-mediated signaling (7): CASP8 forms partially enzymatic...

With the STING C-terminus being extensively studied, the role of the N-terminal domain (NTD) of STING remains an important subject of investigation. Here we identify novel mutations in NTD of Sting of the MOLF strain in response to Herpes Simplex Virus and Listeria monocytogenes both in vitro and in vivo. These mutations are responsible for low levels of IFNβ due to failure of MOLF STING to translocate from the ER. These data provide the first 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.

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.

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