XIAP promotes the expansion and limits the contraction of CD8 T cell response through cell extrinsic and intrinsic mechanisms respectively.

XIAP is an endogenous inhibitor of cell death and inactivating mutations of XIAP are responsible for X-linked lymphoproliferative disease (XLP-2) and primary immunodeficiency, but the mechanism(s) behind these contradictory outcomes have been unclear. We report that during infection of macrophages and dendritic cells with various intracellular bacteria, XIAP restricts cell death and secretion of IL-1ß but promotes increased activation of NFκB and JNK which results in elevated secretion of IL-6 and IL-10. Poor secretion of IL-6 by Xiap-deficient antigen presenting cells leads to poor expansion of recently activated CD8 T cells during the priming phase of the response. On the other hand, Xiap-deficient CD8 T cells displayed increased proliferation and effector function during the priming phase but underwent enhanced contraction subsequently. Xiap-deficient CD8 T cells underwent skewed differentiation towards short lived effectors which resulted in poor generation of memory. Consequently Xiap-deficient CD8 T cells failed to provide effective control of bacterial infection during re-challenge. These results reveal the temporal impact of XIAP in promoting the fitness of activated CD8 T cells through cell extrinsic and intrinsic mechanisms and provide a mechanistic explanation of the phenotype observed in XLP-2 patients.

Divergent SARS-CoV-2 variant emerges in white-tailed deer with deer-to-human transmission.

Wildlife reservoirs of broad-host-range viruses have the potential to enable evolution of viral variants that can emerge to infect humans. In North America, there is phylogenomic evidence of continual transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from humans to white-tailed deer (Odocoileus virginianus) through unknown means, but no evidence of transmission from deer to humans. We carried out an observational surveillance study in Ontario, Canada during November and December 2021 (n = 300 deer) and identified a highly divergent lineage of SARS-CoV-2 in white-tailed deer (B.1.641). This lineage is one of the most divergent SARS-CoV-2 lineages identified so far, with 76 mutations (including 37 previously associated with non-human mammalian hosts). From a set of five complete and two partial deer-derived viral genomes we applied phylogenomic, recombination, selection and mutation spectrum analyses, which provided evidence for evolution and transmission in deer and a shared ancestry with mink-derived virus. Our analysis also revealed an epidemiologically linked human infection. Taken together, our findings provide evidence for sustained evolution of SARS-CoV-2 in white-tailed deer and of deer-to-human transmission.

Identification and differential usage of a host metalloproteinase entry pathway by SARS-CoV-2 Delta and Omicron.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike glycoprotein (S) binds to angiotensin-converting enzyme 2 (ACE2) to mediate membrane fusion via two distinct pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In this study, we found that SARS-CoV-2 S has a wider protease usage and can also be activated by TMPRSS13 and matrix metalloproteinases (MMPs). We found that MMP-2 and MMP-9 played roles in SARS-CoV-2 S cell-cell fusion and TMPRSS2- and cathepsin-independent viral entry in cells expressing high MMP levels. MMP-dependent viral entry required cleavage at the S1/S2 junction in viral producer cells, and differential processing of variants of concern S dictated its usage; the efficiently processed Delta S preferred metalloproteinase-dependent entry when available, and less processed Omicron S was unable to us metalloproteinases for entry. As MMP-2/9 are released during inflammation, they may play roles in S-mediated cytopathic effects, tropism, and disease outcome.

Regional connectivity drove bidirectional transmission of SARS-CoV-2 in the Middle East during travel restrictions.

Regional connectivity and land travel have been identified as important drivers of SARS-CoV-2 transmission. However, the generalizability of this finding is understudied outside of well-sampled, highly connected regions. In this study, we investigated the relative contributions of regional and intercontinental connectivity to the source-sink dynamics of SARS-CoV-2 for Jordan and the Middle East. By integrating genomic, epidemiological and travel data we show that the source of introductions into Jordan was dynamic across 2020, shifting from intercontinental seeding in the early pandemic to more regional seeding for the travel restrictions period. We show that land travel, particularly freight transport, drove introduction risk during the travel restrictions period. High regional connectivity and land travel also drove Jordan's export risk. Our findings emphasize regional connectedness and land travel as drivers of transmission in the Middle East.

Foxo3a tempers excessive glutaminolysis in activated T cells to prevent fatal gut inflammation in the murine IL-10-/- model of colitis.

Mutations in susceptibility alleles correlate with gut-inflammatory diseases, such as Crohn's disease; however, this does not often impact the disease progression indicating the existence of compensatory genes. We show that a reduction in Foxo3a expression in IL-10-deficient mice results in a spontaneous and aggressive Crohn's- like disease with 100% penetrance, which is rescued by deletion of myeloid cells, T cells and inhibition of mTORC1. In Foxo3a-/- IL-10-/- mice, there is poor cell death of myeloid cells in the gut, leading to increased accumulation of myeloid and T cells in the gut. Myeloid cells express high levels of inflammatory cytokines, and regulatory T cells are dysfunctional despite increased abundance. Foxo3a signaling represses the transcription of glutaminase (GLS/GLS2) to prevent over-consumption of glutamine by activated T cells and its conversion to glutamate that contributes to the TCA cycle and mTORC1 activation. Finally, we show that Foxo3a restricts the abundance of colitogenic microbiota in IL-10-deficient mice. Thus, by suppressing glutaminolysis in activated T cells Foxo3a mediates a critical checkpoint that prevents the development of fulminant gut inflammatory disease.

Coating M-CSF on plastic surface results in the generation of increased numbers of macrophages in vitro.

Macrophages are one of the important cell types in the innate immune system that are present in various anatomical regions of the body and promote early control of pathogens. The relative proportion of macrophages in various lymphoid and non-lymphoid regions is small, and as such it is tedious to purify these cells to homogeneity. Culture of bone marrow precursors with macrophage colony-stimulating factor (M-CSF) results in their differentiation to macrophages, however this procedure results in low numbers of differentiated macrophages. Herein we reveal a new approach of generating increased numbers of differentiated macrophages from bone marrow precursors. We show that M-CSF delivered in a plate-bound form results in the differentiation of significantly more macrophages in comparison to soluble M-CSF. Furthermore, the macrophages differentiated with plate-bound M-CSF display increased metabolic activity and cell death following infection with pathogens.

Tristetraprolin regulates necroptosis during tonic Toll-like receptor 4 (TLR4) signaling in murine macrophages.

The necrosome is a protein complex required for signaling in cells that results in necroptosis, which is also dependent on tumor necrosis factor receptor (TNF-R) signaling. TNFα promotes necroptosis, and its expression is facilitated by mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) but is inhibited by the RNA-binding protein tristetraprolin (TTP, encoded by the Zfp36 gene). We have stimulated murine macrophages from WT, MyD88-/-, Trif-/-, MyD88-/-Trif-/-, MK2-/-, and Zfp36-/- mice with graded doses of lipopolysaccharide (LPS) and various inhibitors to evaluate the role of various genes in Toll-like receptor 4 (TLR4)-induced necroptosis. Necrosome signaling, cytokine production, and cell death were evaluated by immunoblotting, ELISA, and cell death assays, respectively. We observed that during TLR4 signaling, necrosome activation is mediated through the adaptor proteins MyD88 and TRIF, and this is inhibited by MK2. In the absence of MK2-mediated necrosome activation, lipopolysaccharide-induced TNFα expression was drastically reduced, but MK2-deficient cells became highly sensitive to necroptosis even at low TNFα levels. In contrast, during tonic TLR4 signaling, WT cells did not undergo necroptosis, even when MK2 was disabled. Of note, necroptosis occurred only in the absence of TTP and was mediated by the expression of TNFα and activation of JUN N-terminal kinase (JNK). These results reveal that TTP plays an important role in inhibiting TNFα/JNK-induced necrosome signaling and resultant cytotoxicity.

Differentiated macrophages acquire a pro-inflammatory and cell death-resistant phenotype due to increasing XIAP and p38-mediated inhibition of RipK1.

Monocytes differentiate into macrophages, which deactivate invading pathogens. Macrophages can be resistant to cell death mechanisms in some situations, and the mechanisms involved are not clear. Here, using mouse immune cells, we investigated whether the differentiation of macrophages affects their susceptibility to cell death by the ripoptosome/necrosome pathways. We show that treatment of macrophages with a mimetic of second mitochondrial activator of caspases (SMAC) resulted in ripoptosome-driven cell death that specifically depended on tumor necrosis factor α (TNFα) expression and the receptor-interacting serine/threonine protein kinase 1 (RipK1)-RipK3-caspase-8 interaction in activated and cycling macrophages. Differentiation of macrophages increased the expression of pro-inflammatory cytokines but reduced RipK1-dependent cell death and the RipK3-caspase-8 interaction. The expression of the anti-apoptotic mediators, X-linked inhibitor of apoptosis protein (XIAP) and caspase-like apoptosis regulatory protein (cFLIPL), also increased in differentiated macrophages, which inhibited caspase activation. The resistance to cell death was abrogated in XIAP-deficient macrophages. However, even in the presence of increased XIAP expression, inhibition of the mitogen-activated protein kinase (MAPK) p38 and MAPK-activated protein kinase 2 (MK2) made differentiated macrophages susceptible to cell death. These results suggest that the p38/MK2 pathway overrides apoptosis inhibition by XIAP and that acquisition of resistance to cell death by increased expression of XIAP and cFLIPL may allow inflammatory macrophages to participate in pathogen control for a longer duration.

Triad3a induces the degradation of early necrosome to limit RipK1-dependent cytokine production and necroptosis.

Understanding the molecular signaling in programmed cell death is vital to a practical understanding of inflammation and immune cell function. Here we identify a previously unrecognized mechanism that functions to downregulate the necrosome, a central signaling complex involved in inflammation and necroptosis. We show that RipK1 associates with RipK3 in an early necrosome, independent of RipK3 phosphorylation and MLKL-induced necroptotic death. We find that formation of the early necrosome activates K48-ubiquitin-dependent proteasomal degradation of RipK1, Caspase-8, and other necrosomal proteins. Our results reveal that the E3-ubiquitin ligase Triad3a promotes this negative feedback loop independently of typical RipK1 ubiquitin editing enzymes, cIAPs, A20, or CYLD. Finally, we show that Triad3a-dependent necrosomal degradation limits necroptosis and production of inflammatory cytokines. These results reveal a new mechanism of shutting off necrosome signaling and may pave the way to new strategies for therapeutic manipulation of inflammatory responses.

Genetic variation in populations of Allothrombium pulvinum (Acari: Trombidiidae) from Northern Iran revealed by mitochondrial coxI and nuclear rDNA ITS2 sequences.

Allothrombium pulvinum Ewing is a common natural enemy of aphids and some other arthropods. So far, there are no studies that have addressed genetic variation of this predatory mite. We investigated genetic variation of A. pulvinum across its whole known range in Iran. A 410 bp portion of the mitochondrial cytochrome c oxidase subunit I gene (coxI) and 797-802 bp portion of the internal transcribed spacer 2 of rDNA (ITS2) were sequenced for 55 individuals from 11 populations, resulting in 12 and 26 haplotypes, respectively. In the coxI region, haplotype and nucleotide diversities varied among populations from 0.00 to 0.90 and from 0.0000 to 0.0110, respectively. In the ITS2 region they varied from 0.20 to 0.91 and from 0.0006 to 0.0023, respectively. For both gene regions the highest haplotype and nucleotide diversities were detected in population Mahmoud Abad from northern Iran. Statistically significant population differentiation (F (ST)) was detected in most pair-wise population comparisons. The results of population differentiation for both gene regions were generally congruent indicating that A. pulvinum from Iran consists of genetically different populations. This suggests that A. pulvinum comprises at least two geographically distinct populations or even more than one species. This study is an initial step towards understanding genetic variation of A. pulvinum, a taxon for which little molecular information is available. More intensive sampling and analysis of additional DNA regions are necessary for more detailed classification of this taxon.

Laboratory evaluation of some plant essences to control Varroa destructor (Acari: Varroidae).

This research was conducted to evaluate acaricidal effects of some plant essences on Varroa mites and the possibility of their usage for Varroa control. First, live Varroa mites were obtained from adult honeybees with CO2 in a newly designed apparatus. Thyme, savory, rosemary, marjoram, dillsun and lavender essences at concentrations of 2 and 1 g/100 g (w/w), caused a mite mortality rate of more than 97% and 95%, respectively. Also spearmint at 2 g/100 g was able to kill more than 97% of Varroa mites. When sprayed on worker honeybees infected with mites, thyme, savory, spearmint and dillsun essences at 2 g/100 g (w/w) caused 43-58% Varroa mortality. Toxicity of thyme, savory and spearmint essences for worker honeybees was not significantly different from that of controls (acetone and water), but dillsun essence caused 12% honeybee mortality. These results showed that essences of thyme, savory and spearmint have acaricidal properties that could be used for controlling Varroa in honeybee colonies.