Threonine phosphorylation of STAT1 restricts interferon signaling and promotes innate inflammatory responses.

Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and antiviral defense. Here, using genetic and biochemical assays, we unveil Thr748 as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically engineered mice expressing phospho-deficient threonine748-to-alanine (T748A) mutant Stat1 are resistant to LPS-induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of Stat1 following LPS-induced lethality, indicating that the Thr748 phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic IκB kinase-mediated Thr748 phosphorylation of Stat1, which promotes macrophage inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent modular functionality of Stat1 in innate immune responses: IFN phospho-tyrosine dependent and inflammatory phospho-threonine dependent. Better understanding of the Thr748 phosphorylation of Stat1 may uncover advanced pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually.

Systemic inflammatory response syndrome triggered by blood-borne pathogens induces prolonged dendritic cell paralysis and immunosuppression.

Blood-borne pathogens can cause systemic inflammatory response syndrome (SIRS) followed by protracted, potentially lethal immunosuppression. The mechanisms responsible for impaired immunity post-SIRS remain unclear. We show that SIRS triggered by pathogen mimics or malaria infection leads to functional paralysis of conventional dendritic cells (cDCs). Paralysis affects several generations of cDCs and impairs immunity for 3-4 weeks. Paralyzed cDCs display distinct transcriptomic and phenotypic signatures and show impaired capacity to capture and present antigens in vivo. They also display altered cytokine production patterns upon stimulation. The paralysis program is not initiated in the bone marrow but during final cDC differentiation in peripheral tissues under the influence of local secondary signals that persist after resolution of SIRS. Vaccination with monoclonal antibodies that target cDC receptors or blockade of transforming growth factor ß partially overcomes paralysis and immunosuppression. This work provides insights into the mechanisms of paralysis and describes strategies to restore immunocompetence post-SIRS.

Antigen-inexperienced memory CD8+ T cells: where they come from and why we need them.

Memory-phenotype CD8+ T cells exist in substantial numbers within hosts that have not been exposed to either foreign antigen or overt lymphopenia. These antigen-inexperienced memory-phenotype T cells can be divided into two major subsets: 'innate memory' T cells and 'virtual memory' T cells. Although these two subsets are nearly indistinguishable by surface markers alone, notable developmental and functional differences exist between the two subsets, which suggests that they represent distinct populations. In this Opinion article, we review the available literature on each subset, highlighting the key differences between these populations. Furthermore, we suggest a unifying model for the categorization of antigen-inexperienced memory-phenotype CD8+ T cells.

Virtual memory T cells develop and mediate bystander protective immunity in an IL-15-dependent manner.

Virtual memory cells (VM) are an antigen-specific, memory phenotype CD8 T-cell subset found in lymphoreplete, unchallenged mice. Previous studies indicated that VM cells were the result of homeostatic proliferation (HP) resembling the proliferation observed in a lymphopenic environment. Here we demonstrate that HP is ongoing in lymphoreplete mice, the degree of which is dictated by the number of naive CD8 T cells with a sufficiently high affinity for self-antigen interacting with peripheral IL-15. VM cell transcriptional profiles suggest a capacity to mediate protective immunity via antigen non-specific bystander killing, a function we show is dependent on IL-15. Finally, we show a VM-like population of human cells that accumulate with age and traffic to the liver, displaying phenotypic and functional attributes consistent with the bystander protective functions of VM cells identified in the mouse. These data identify developmental and functional attributes of VM cells, including their likely role in protective immunity.

CD8α+ dendritic cell trans presentation of IL-15 to naive CD8+ T cells produces antigen-inexperienced T cells in the periphery with memory phenotype and function.

Various populations of memory phenotype CD8(+) T cells have been described over the last 15-20 y, all of which possess elevated effector functions relative to naive phenotype cells. Using a technique for isolating Ag-specific cells from unprimed hosts, we recently identified a new subset of cells, specific for nominal Ag, but phenotypically and functionally similar to memory cells arising as a result of homeostatic proliferation. We show in this study that these virtual memory (VM) cells are independent of previously identified innate memory cells, arising as a result of their response to IL-15 trans presentation by lymphoid tissue-resident CD8α(+) dendritic cells in the periphery. The absence of IL-15, CD8(+) T cell expression of either CD122 or eomesodermin or of CD8a(+) dendritic cells all lead to the loss of VM cells in the host. Our results show that CD8(+) T cell homeostatic expansion is an active process within the nonlymphopenic environment, is mediated by IL-15, and produces Ag-inexperienced memory cells that retain the capacity to respond to nominal Ag with memory-like function. Preferential engagement of these VM T cells into a vaccine response could dramatically enhance the rate by which immune protection develops.

T cell vaccinology: exploring the known unknowns.

The objective of modern vaccine development is the safe generation of protective long-term immune memory, both prophylactic and therapeutic. Live attenuated vaccines generate potent cellular and humoral immunity [1-3], but numerous problems exist with these vaccines, ranging from production and storage issues to adverse reactions and reversion to virulence. Subunit vaccines are safer, more stable, and more amenable to mass production. However the protection they produce is frequently inferior to live attenuated vaccines and is typically confined to humoral, and not cellular immunity. Unfortunately, there are presently no subunit vaccines available clinically that are effective at eliciting cellular responses let alone cellular memory [4]. This article will provide and overview of areas of investigation that we see as important for the development of vaccines with the capacity to induce robust and enduring cellular immune responses.

Role of C3a receptors, C5a receptors, and complement protein C6 deficiency in collagen antibody-induced arthritis in mice.

The complement system, especially the alternative pathway, plays essential roles in the induction of injury in collagen Ab-induced arthritis (CAIA) in mice. The goal of the current study was to directly compare the roles of receptors for C3a and C5a, as well as the membrane attack complex, as effector mechanisms in the pathogenesis of CAIA. Clinical disease activity in C3aR(-/-), C5aR(-/-), and C6-deficient (C6-def) mice was decreased by 52, 94, and 65%, respectively, as compared with wild-type mice. Decreases in histopathologic injury as well as in IgG and C3 deposition paralleled the clinical disease activity. A decrease in the percentage of synovial neutrophils was observed in C3aR(-/-), C5aR(-/-), and C6-def mice, and a decrease in macrophages was observed in C3aR(-/-) and C5aR(-/-), but not in C6-def, mice. Synovial mRNA obtained by laser capture microdissection exhibited a decrease in TNF-α in C5aR(-/-) mice and in IL-1ß in both C5aR(-/-) and C6-def mice, whereas C3aR(-/-) mice demonstrated no change in either cytokine. Our findings show that absent C3aR-, C5aR-, or membrane attack complex-initiated effector mechanisms each decrease susceptibility to CAIA, with clinical effects most pronounced in C5aR-deficient mice. Although the absence of C3aR, C5aR, or C6 led to differential deficiencies in effector mechanisms, decreased proximal joint IgG and C3 deposition was common to all three genotypes in comparison with wild-type mice. These data suggest the existence of positive-feedback amplification pathways downstream of all three effectors that promote additional IgG deposition and C3 activation in the joint.

Statins decrease chondroitin sulfate proteoglycan expression and acute astrocyte activation in central nervous system injury.

Statins elicit numerous favorable effects on central nervous system (CNS) injury, including inhibition of the rhoA/ROCK pathway. In the present study, we show that statins decrease acute astrocyte activation in CNS injury, and decrease chondroitin sulfate proteoglycan (CSPG) levels in astrocyte cultures as well as CNS injury. CSPG levels decreased by up to 45% in simvastatin-treated astrocyte cultures compared to control cultures. In simvastatin-treated animals, CSPG levels declined by 60% 8 days after brain stab injury, and by 62-64% 4 weeks after spinal cord injury (SCI). Glial fibrillary acid protein (GFAP) levels decreased in brain stab at 8 days after surgery/intervention, suggesting that statins produce a decrease in astrocyte activation. Attenuation of astrocyte activation may contribute to the decline in CSPG levels. However, there are likely other contributing factors, since GFAP levels were not a contributing factor in the decline of CSPG levels in astrocyte cultures. Robust locomotor improvements were not observed with any treatment. The numerous beneficial effects of statins on CNS injury render them an attractive candidate in the treatment of CNS injury.