An increase in glycoprotein concentration on extracellular virions dramatically alters vaccinia virus infectivity and pathogenesis without impacting immunogenicity.

The extracellular virion (EV) form of Orthopoxviruses is required for cell-to-cell spread and pathogenesis, and is the target of neutralizing antibodies in the protective immune response. EV have a double envelope that contains several unique proteins that are involved in its intracellular envelopment and/or subsequent infectivity. One of these, F13, is involved in both EV formation and infectivity. Here, we report that replacement of vaccinia virus F13L with the molluscum contagiosum virus homolog, MC021L, results in the production of EV particles with significantly increased levels of EV glycoproteins, which correlate with a small plaque phenotype. Using a novel fluorescence-activated virion sorting assay to isolate EV populations based on glycoprotein content we determine that EV containing either higher or lower levels of glycoproteins are less infectious, suggesting that there is an optimal concentration of glycoproteins in the outer envelope that is required for maximal infectivity of EV. This optimal glycoprotein concentration was required for lethality and induction of pathology in a cutaneous model of animal infection, but was not required for induction of a protective immune response. Therefore, our results demonstrate that there is a sensitive balance between glycoprotein incorporation, infectivity, and pathogenesis, and that manipulation of EV glycoprotein levels can produce vaccine vectors in which pathologic side effects are attenuated without a marked diminution in induction of protective immunity.

IL-4 Treatment Mitigates Experimental Cerebral Malaria by Reducing Parasitemia, Dampening Inflammation, and Lessening the Cytotoxicity of T Cells.

Cytokine responses to malaria play important roles in both protective immunity development and pathogenesis. Although the roles of cytokines such as TNF-α, IL-12, IFN-γ, and IL-10 in immunity and pathogenesis to the blood stage malaria are largely known, the role of IL-4 remains less understood. IL-4 targets many cell types and induces multiple effects, including cell proliferation, gene expression, protection from apoptosis, and immune regulation. Accordingly, IL-4 has been exploited as a therapeutic for several inflammatory diseases. Malaria caused by Plasmodium falciparum manifests in many organ-specific fatal pathologies, including cerebral malaria (CM), driven by a high parasite load, leading to parasite sequestration in organs and consequent excessive inflammatory responses and endothelial damage. We investigated the therapeutic potential of IL-4 against fatal malaria in Plasmodium berghei ANKA-infected C57BL/6J mice, an experimental CM model. IL-4 treatment significantly reduced parasitemia, CM pathology, and mortality. The therapeutic effect of IL-4 is mediated through multiple mechanisms, including enhanced parasite clearance mediated by upregulation of phagocytic receptors and increased IgM production, and decreased brain inflammatory responses, including reduced chemokine (CXCL10) production, reduced chemokine receptor (CXCR3) and adhesion molecule (LFA-1) expression by T cells, and downregulation of cytotoxic T cell lytic potential. IL-4 treatment markedly reduced the infiltration of CD8+ T cells and brain pathology. STAT6, PI3K-Akt-NF-κB, and Src signaling mediated the cellular and molecular events that contributed to the IL-4-dependent decrease in parasitemia. Overall, our results provide mechanistic insights into how IL-4 treatment mitigates experimental CM and have implications in developing treatment strategies for organ-specific fatal malaria.

IL-4Rα signaling by CD8α+ dendritic cells contributes to cerebral malaria by enhancing inflammatory, Th1, and cytotoxic CD8+ T cell responses.

Persistent high levels of proinflammatory and Th1 responses contribute to cerebral malaria (CM). Suppression of inflammatory responses and promotion of Th2 responses prevent pathogenesis. IL-4 commonly promotes Th2 responses and inhibits inflammatory and Th1 responses. Therefore, IL-4 is widely considered as a beneficial cytokine via its Th2-promoting role that is predicted to provide protection against severe malaria by inhibiting inflammatory responses. However, IL-4 may also induce inflammatory responses, as the result of IL-4 action depends on the timing and levels of its production and the tissue environment in which it is produced. Recently, we showed that dendritic cells (DCs) produce IL-4 early during malaria infection in response to a parasite protein and that this IL-4 response may contribute to severe malaria. However, the mechanism by which IL-4 produced by DCs contributing to lethal malaria is unknown. Using Plasmodium berghei ANKA-infected C57BL/6 mice, a CM model, we show here that mice lacking IL-4Rα only in CD8α+ DCs are protected against CM pathogenesis and survive, whereas WT mice develop CM and die. Compared with WT mice, mice lacking IL-4Rα in CD11c+ or CD8α+ DCs showed reduced inflammatory responses leading to decreased Th1 and cytotoxic CD8+ T cell responses, lower infiltration of CD8+ T cells to the brain, and negligible brain pathology. The novel results presented here reveal a paradoxical role of IL-4Rα signaling in CM pathogenesis that promotes CD8α+ DC-mediated inflammatory responses that generate damaging Th1 and cytotoxic CD8+ T cell responses.

Vaccinia Virus Glycoproteins A33, A34, and B5 Form a Complex for Efficient Endoplasmic Reticulum to trans-Golgi Network Transport.

Orthopoxviruses produce two, antigenically distinct, infectious enveloped virions termed intracellular mature virions and extracellular virions. Extracellular virions are required for cell-to-cell spread and pathogenesis. Specific to the extracellular virion membrane, glycoproteins A33, A34, and B5 are highly conserved among orthopoxviruses and have roles during extracellular virion formation and subsequent infection. B5 is dependent on an interaction with either A33 or A34 for localization to the site of intracellular envelopment and incorporation into the envelope of released extracellular virions. In this report we show that an interaction between A33 and A34 can be detected in infected cells. Furthermore, we show that a three-protein complex between A33, A34, and B5 forms in the endoplasmic reticulum (ER) that disassociates post ER export. Finally, immunofluorescence reveals that coexpression of all three glycoproteins results in their localization to a juxtanuclear region that is presumably the site of intracellular envelopment. These results demonstrate the existence of two previously unidentified interactions: one between A33 and A34 and another simultaneous interaction between all three of the glycoproteins. Furthermore, these results indicate that interactions among A33, A34, and B5 are vital for proper intracellular trafficking and subcellular localization.IMPORTANCE The secondary intracellular envelopment of poxviruses at the trans-Golgi network to release infectious extracellular virus (EV) is essential for their spread and pathogenesis. Viral glycoproteins A33, A34, and B5 are critical for the efficient production of infectious EV and interactions among these proteins are important for their localization and incorporation into the outer extracellular virion membrane. We have uncovered a novel interaction between glycoproteins A33 and A34. Furthermore, we show that B5 can interact with the A33-A34 complex. Our analysis indicates that the three-protein complex has a role in ER exit and proper localization of the three glycoproteins to the intracellular site of wrapping. These results show that a complex set of interactions occur in the secretory pathway of infected cells to ensure proper glycoprotein trafficking and envelope content, which is important for the release of infectious poxvirus virions.

Type I interferon-dependent CCL4 is induced by a cGAS/STING pathway that bypasses viral inhibition and protects infected tissue, independent of viral burden.

Type I interferons (T1-IFN) are critical in the innate immune response, acting upon infected and uninfected cells to initiate an antiviral state by expressing genes that inhibit multiple stages of the lifecycle of many viruses. T1-IFN triggers the production of Interferon-Stimulated Genes (ISGs), activating an antiviral program that reduces virus replication. The importance of the T1-IFN response is highlighted by the evolution of viral evasion strategies to inhibit the production or action of T1-IFN in virus-infected cells. T1-IFN is produced via activation of pathogen sensors within infected cells, a process that is targeted by virus-encoded immunomodulatory molecules. This is probably best exemplified by the prototypic poxvirus, Vaccinia virus (VACV), which uses at least 6 different mechanisms to completely block the production of T1-IFN within infected cells in vitro. Yet, mice lacking aspects of T1-IFN signaling are often more susceptible to infection with many viruses, including VACV, than wild-type mice. How can these opposing findings be rationalized? The cytosolic DNA sensor cGAS has been implicated in immunity to VACV, but has yet to be linked to the production of T1-IFN in response to VACV infection. Indeed, there are two VACV-encoded proteins that effectively prevent cGAS-mediated activation of T1-IFN. We find that the majority of VACV-infected cells in vivo do not produce T1-IFN, but that a small subset of VACV-infected cells in vivo utilize cGAS to sense VACV and produce T1-IFN to protect infected mice. The protective effect of T1-IFN is not mediated via ISG-mediated control of virus replication. Rather, T1-IFN drives increased expression of CCL4, which recruits inflammatory monocytes that constrain the VACV lesion in a virus replication-independent manner by limiting spread within the tissue. Our findings have broad implications in our understanding of pathogen detection and viral evasion in vivo, and highlight a novel immune strategy to protect infected tissue.

A systemic macrophage response is required to contain a peripheral poxvirus infection.

The goal of the innate immune system is to reduce pathogen spread prior to the initiation of an effective adaptive immune response. Following an infection at a peripheral site, virus typically drains through the lymph to the lymph node prior to entering the blood stream and being systemically disseminated. Therefore, there are three distinct spatial checkpoints at which intervention to prevent systemic spread of virus can occur, namely: 1) the site of infection, 2) the draining lymph node via filtration of lymph or 3) the systemic level via organs that filter the blood. We have previously shown that systemic depletion of phagocytic cells allows viral spread after dermal infection with Vaccinia virus (VACV), which infects naturally through the skin. Here we use multiple depletion methodologies to define both the spatial checkpoint and the identity of the cells that prevent systemic spread of VACV. Subcapsular sinus macrophages of the draining lymph node have been implicated as critical effectors in clearance of lymph borne viruses following peripheral infection. We find that monocyte populations recruited to the site of VACV infection play a critical role in control of local pathogenesis and tissue damage, but do not prevent dissemination of virus. Following infection with virulent VACV, the subcapsular sinus macrophages within the draining lymph node become infected, but are not exclusively required to prevent systemic spread. Rather, small doses of VACV enter the bloodstream and the function of systemic macrophages, but not dendritic cells, is required to prevent further spread. The results illustrate that a systemic innate response to a peripheral virus infection may be required to prevent widespread infection and pathology following infection with virulent viruses, such as poxviruses.

Small molecule-based inhibition of MEK1/2 proteins dampens inflammatory responses to malaria, reduces parasite load, and mitigates pathogenic outcomes.

Malaria infections cause several systemic and severe single- or multi-organ pathologies, killing hundreds of thousands of people annually. Considering the existing widespread resistance of malaria parasites to anti-parasitic drugs and their high propensity to develop drug resistance, alternative strategies are required to manage malaria infections. Because malaria is a host immune response-driven disease, one approach is based on gaining a detailed understanding of the molecular and cellular processes that modulate malaria-induced innate and adaptive immune responses. Here, using a mouse cerebral malaria model and small-molecule inhibitors, we demonstrate that inhibiting MEK1/2, the upstream kinases of ERK1/2 signaling, alters multifactorial components of the innate and adaptive immune responses, controls parasitemia, and blocks pathogenesis. Specifically, MEK1/2 inhibitor treatment up-regulated B1 cell expansion, IgM production, phagocytic receptor expression, and phagocytic activity, enhancing parasite clearance by macrophages and neutrophils. Further, the MEK1/2 inhibitor treatment down-regulated pathogenic pro-inflammatory and helper T cell 1 (Th1) responses and up-regulated beneficial anti-inflammatory cytokine responses and Th2 responses. These inhibitor effects resulted in reduced granzyme B expression by T cells, chemokine and intracellular cell adhesion molecule 1 (ICAM-1) expression in the brain, and chemokine receptor expression by both myeloid and T cells. These bimodal effects of the MEK1/2 inhibitor treatment on immune responses contributed to decreased parasite biomass, organ inflammation, and immune cell recruitment, preventing tissue damage and death. In summary, we have identified several previously unrecognized immune regulatory processes through which a MEK1/2 inhibitor approach controls malaria parasitemia and mitigates pathogenic effects on host organs.

Peptide-MHC-I from Endogenous Antigen Outnumber Those from Exogenous Antigen, Irrespective of APC Phenotype or Activation.

Naïve anti-viral CD8+ T cells (TCD8+) are activated by the presence of peptide-MHC Class I complexes (pMHC-I) on the surface of professional antigen presenting cells (pAPC). Increasing the number of pMHC-I in vivo can increase the number of responding TCD8+. Antigen can be presented directly or indirectly (cross presentation) from virus-infected and uninfected cells, respectively. Here we determined the relative importance of these two antigen presenting pathways in mousepox, a natural disease of the mouse caused by the poxvirus, ectromelia (ECTV). We demonstrated that ECTV infected several pAPC types (macrophages, B cells, and dendritic cells (DC), including DC subsets), which directly presented pMHC-I to naïve TCD8+ with similar efficiencies in vitro. We also provided evidence that these same cell-types presented antigen in vivo, as they form contacts with antigen-specific TCD8+. Importantly, the number of pMHC-I on infected pAPC (direct presentation) vastly outnumbered those on uninfected cells (cross presentation), where presentation only occurred in a specialized subset of DC. In addition, prior maturation of DC failed to enhance antigen presentation, but markedly inhibited ECTV infection of DC. These results suggest that direct antigen presentation is the dominant pathway in mice during mousepox. In a broader context, these findings indicate that if a virus infects a pAPC then the presentation by that cell is likely to dominate over cross presentation as the most effective mode of generating large quantities of pMHC-I is on the surface of pAPC that endogenously express antigens. Recent trends in vaccine design have focused upon the introduction of exogenous antigens into the MHC Class I processing pathway (cross presentation) in specific pAPC populations. However, use of a pantropic viral vector that targets pAPC to express antigen endogenously likely represents a more effective vaccine strategy than the targeting of exogenous antigen to a limiting pAPC subpopulation.

Redundant Function of Plasmacytoid and Conventional Dendritic Cells Is Required To Survive a Natural Virus Infection.

UNLABELLED: Viruses that spread systemically from a peripheral site of infection cause morbidity and mortality in the human population. Innate myeloid cells, including monocytes, macrophages, monocyte-derived dendritic cells (mo-DC), and dendritic cells (DC), respond early during viral infection to control viral replication, reducing virus spread from the peripheral site. Ectromelia virus (ECTV), an orthopoxvirus that naturally infects the mouse, spreads systemically from the peripheral site of infection and results in death of susceptible mice. While phagocytic cells have a requisite role in the response to ECTV, the requirement for individual myeloid cell populations during acute immune responses to peripheral viral infection is unclear. In this study, a variety of myeloid-specific depletion methods were used to dissect the roles of individual myeloid cell subsets in the survival of ECTV infection. We showed that DC are the primary producers of type I interferons (T1-IFN), requisite cytokines for survival, following ECTV infection. DC, but not macrophages, monocytes, or granulocytes, were required for control of the virus and survival of mice following ECTV infection. Depletion of either plasmacytoid DC (pDC) alone or the lymphoid-resident DC subset (CD8α(+) DC) alone did not confer lethal susceptibility to ECTV. However, the function of at least one of the pDC or CD8α(+) DC subsets is required for survival of ECTV infection, as mice depleted of both populations were susceptible to ECTV challenge. The presence of at least one of these DC subsets is sufficient for cytokine production that reduces ECTV replication and virus spread, facilitating survival following infection. IMPORTANCE: Prior to the eradication of variola virus, the orthopoxvirus that causes smallpox, one-third of infected people succumbed to the disease. Following successful eradication of smallpox, vaccination rates with the smallpox vaccine have significantly dropped. There is now an increasing incidence of zoonotic orthopoxvirus infections for which there are no effective treatments. Moreover, the safety of the smallpox vaccine is of great concern, as complications may arise, resulting in morbidity. Like many viruses that cause significant human diseases, orthopoxviruses spread from a peripheral site of infection to become systemic. This study elucidates the early requirement for innate immune cells in controlling a peripheral infection with ECTV, the causative agent of mousepox. We report that there is redundancy in the function of two innate immune cell subsets in controlling virus spread early during infection. The viral control mediated by these cell subsets presents a potential target for therapies and rational vaccine design.

Prolonged antigen presentation following an acute virus infection requires direct and then cross-presentation.

Antiviral CD8(+) T cell recognition of MHC class I-peptide complexes on the surface of professional APCs is a requisite step in an effective immune response following many potentially lethal infections. Although MHC class I-peptide production is thought to be closely linked to the continued presence of virus, several studies have shown that the persistence of Ag presentation occurs for an extended period of time following the clearance of RNA viruses. However, the mechanism responsible for Ag presentation persistence following viral clearance was unknown until now. In this study, we used a recombinant DNA virus expressing different forms of a model Ag to study the mechanism of prolonged Ag presentation in mice. We determined that the persistence of Ag presentation consists of three distinct mechanistic phases, as follows: ongoing viral replication, persistence of virally infected cells, and cross-presentation of Ag. These data will allow manipulation of the form of Ag contained within viral vectors to produce the most effective and protective CD8(+) T cell response to be generated following vaccination.

MyD88-dependent immunity to a natural model of vaccinia virus infection does not involve Toll-like receptor 2.

UNLABELLED: Although the pattern recognition receptor Toll-like receptor 2 (TLR2) is typically thought to recognize bacterial components, it has been described to alter the induction of both innate and adaptive immunity to a number of viruses, including vaccinia virus (VACV). However, many pathogens that reportedly encode TLR2 agonists may actually be artifactually contaminated during preparation, possibly with cellular debris or merely with molecules that sensitize cells to be activated by authentic TLR2 agonists. In both humans and mice, the most relevant natural route of infection with VACV is through intradermal infection of the skin. Therefore, we examined the requirement for TLR2 and its signaling adaptor MyD88 in protective immunity to VACV after intradermal infection. We find that although TLR2 may recognize virus preparations in vitro and have a minor role in preventing dissemination of VACV following systemic infection with large doses of virus, it is wholly disposable in both control of virus replication and induction of adaptive immunity following intradermal infection. In contrast, MyD88 is required for efficient induction of CD4 T cell and B cell responses and for local control of virus replication following intradermal infection. However, even MyD88 is not required to induce local inflammation, inflammatory cytokine production, or recruitment of cells that restrict virus from spreading systemically after peripheral infection. Thus, an effective antiviral response does require MyD88, but TLR2 is not required for control of a peripheral VACV infection. These findings emphasize the importance of studying relevant routes of infection when examining innate sensing mechanisms. IMPORTANCE: Vaccinia virus (VACV) provides the backbone for some of the most widely used and successful viral vaccine vectors and is also related to the human pathogens Cantagalo virus and molluscum contagiosum virus that infect the skin of patients. Therefore, it is vital to understand the mechanisms that induce a strong innate immune response to the virus following dermal infection. Here, we compare the ability of the innate sensing molecule Toll-like receptor 2 (TLR2) and the signaling molecule MyD88 to influence the innate and adaptive immune response to VACV following systemic or dermal infection.

Antibody inhibition of a viral type 1 interferon decoy receptor cures a viral disease by restoring interferon signaling in the liver.

Type 1 interferons (T1-IFNs) play a major role in antiviral defense, but when or how they protect during infections that spread through the lympho-hematogenous route is not known. Orthopoxviruses, including those that produce smallpox and mousepox, spread lympho-hematogenously. They also encode a decoy receptor for T1-IFN, the T1-IFN binding protein (T1-IFNbp), which is essential for virulence. We demonstrate that during mousepox, T1-IFNs protect the liver locally rather than systemically, and that the T1-IFNbp attaches to uninfected cells surrounding infected foci in the liver and the spleen to impair their ability to receive T1-IFN signaling, thus facilitating virus spread. Remarkably, this process can be reversed and mousepox cured late in infection by treating with antibodies that block the biological function of the T1-IFNbp. Thus, our findings provide insights on how T1-IFNs function and are evaded during a viral infection in vivo, and unveil a novel mechanism for antibody-mediated antiviral therapy.

Local activation of dendritic cells alters the pathogenesis of autoimmune disease in the retina.

Interest in the identities, properties, functions, and origins of local APC in CNS tissues is growing. We recently reported that dendritic cells (DC) distinct from microglia were present in quiescent retina and rapidly responded to injured neurons. In this study, the disease-promoting and regulatory contributions of these APC in experimental autoimmune uveoretinitis (EAU) were examined. Local delivery of purified, exogenous DC or monocytes from bone marrow substantially increased the incidence and severity of EAU induced by adoptive transfer of activated, autoreactive CD4 or CD8 T cells that was limited to the manipulated eye. In vitro assays of APC activity of DC from quiescent retina showed that they promoted generation of Foxp3(+) T cells and inhibited activation of naive T cells by splenic DC and Ag. Conversely, in vitro assays of DC purified from injured retina demonstrated an enhanced ability to activate T cells and reduced induction of Foxp3(+) T cells. These findings were supported by the observation that in situ activation of DC before adoptive transfer of ß-galactosidase-specific T cells dramatically increased severity and incidence of EAU. Recruitment of T cells into retina by local delivery of Ag in vivo showed that quiescent retina promoted development of parenchymal Foxp3(+) T cells, but assays of preinjured retina did not. Together, these results demonstrated that local conditions in the retina determined APC function and affected the pathogenesis of EAU by both CD4 and CD8 T cells.

γδ T Cells Mediate a Requisite Portion of a Wound Healing Response Triggered by Cutaneous Poxvirus Infection.

Infection at barrier sites, e.g., skin, activates local immune defenses that limit pathogen spread, while preserving tissue integrity. Phenotypically distinct γδ T cell populations reside in skin, where they shape immunity to cutaneous infection prior to onset of an adaptive immune response by conventional αß CD4+ (TCD4+) and CD8+ (TCD8+) T cells. To examine the mechanisms used by γδ T cells to control cutaneous virus replication and tissue pathology, we examined γδ T cells after infection with vaccinia virus (VACV). Resident γδ T cells expanded and combined with recruited γδ T cells to control pathology after VACV infection. However, γδ T cells did not play a role in control of local virus replication or blockade of systemic virus spread. We identified a unique wound healing signature that has features common to, but also features that antagonize, the sterile cutaneous wound healing response. Tissue repair generally occurs after clearance of a pathogen, but viral wound healing started prior to the peak of virus replication in the skin. γδ T cells contributed to wound healing through induction of multiple cytokines/growth factors required for efficient wound closure. Therefore, γδ T cells modulate the wound healing response following cutaneous virus infection, maintaining skin barrier function to prevent secondary bacterial infection.

Deubiquitinating enzyme CYLD negatively regulates the ubiquitin-dependent kinase Tak1 and prevents abnormal T cell responses.

The deubiquitinating enzyme CYLD has recently been implicated in the regulation of signal transduction, but its physiological function and mechanism of action are still elusive. In this study, we show that CYLD plays a pivotal role in regulating T cell activation and homeostasis. T cells derived from Cyld knockout mice display a hyperresponsive phenotype and mediate the spontaneous development of intestinal inflammation. Interestingly, CYLD targets a ubiquitin-dependent kinase, transforming growth factor-beta-activated kinase 1 (Tak1), and inhibits its ubiquitination and autoactivation. Cyld-deficient T cells exhibit constitutively active Tak1 and its downstream kinases c-Jun N-terminal kinase and IkappaB kinase beta. These results emphasize a critical role for CYLD in preventing spontaneous activation of the Tak1 axis of T cell signaling and, thereby, maintaining normal T cell function.

CD11b⁺, Ly6G⁺ cells produce type I interferon and exhibit tissue protective properties following peripheral virus infection.

The goal of the innate immune system is containment of a pathogen at the site of infection prior to the initiation of an effective adaptive immune response. However, effector mechanisms must be kept in check to combat the pathogen while simultaneously limiting undesirable destruction of tissue resulting from these actions. Here we demonstrate that innate immune effector cells contain a peripheral poxvirus infection, preventing systemic spread of the virus. These innate immune effector cells are comprised primarily of CD11b⁺Ly6C⁺Ly6G⁻ monocytes that accumulate initially at the site of infection, and are then supplemented and eventually replaced by CD11b⁺Ly6C⁺Ly6G⁺ cells. The phenotype of the CD11b⁺Ly6C⁺Ly6G⁺ cells resembles neutrophils, but the infiltration of neutrophils typically occurs prior to, rather than following, accumulation of monocytes. Indeed, it appears that the CD11b⁺Ly6C⁺Ly6G⁺ cells that infiltrated the site of VACV infection in the ear are phenotypically distinct from the classical description of both neutrophils and monocyte/macrophages. We found that CD11b⁺Ly6C⁺Ly6G⁺ cells produce Type I interferons and large quantities of reactive oxygen species. We also observed that depletion of Ly6G⁺ cells results in a dramatic increase in tissue damage at the site of infection. Tissue damage is also increased in the absence of reactive oxygen species, although reactive oxygen species are typically thought to be damaging to tissue rather than protective. These data indicate the existence of a specialized population of CD11b⁺Ly6C⁺Ly6G⁺ cells that infiltrates a site of virus infection late and protects the infected tissue from immune-mediated damage via production of reactive oxygen species. Regulation of the action of this population of cells may provide an intervention to prevent innate immune-mediated tissue destruction.

A marked reduction in priming of cytotoxic CD8+ T cells mediated by stress-induced glucocorticoids involves multiple deficiencies in cross-presentation by dendritic cells.

Protracted psychological stress elevates circulating glucocorticoids, which can suppress CD8(+) T cell-mediated immunity, but the mechanisms are incompletely understood. Dendritic cells (DCs), required for initiating CTL responses, are vulnerable to stress/corticosterone, which can contribute to diminished CTL responses. Cross-priming of CD8(+) T cells by DCs is required for initiating CTL responses against many intracellular pathogens that do not infect DCs. We examined the effects of stress/corticosterone on MHC class I (MHC I) cross-presentation and priming and show that stress/corticosterone-exposed DCs have a reduced ability to cross-present OVA and activate MHC I-OVA(257-264)-specific T cells. Using a murine model of psychological stress and OVA-loaded ß(2)-microglobulin knockout "donor" cells that cannot present Ag, DCs from stressed mice induced markedly less Ag-specific CTL proliferation in a glucocorticoid receptor-dependent manner, and endogenous in vivo T cell cytolytic activity generated by cross-presented Ag was greatly diminished. These deficits in cross-presentation/priming were not due to altered Ag donation, Ag uptake (phagocytosis, receptor-mediated endocytosis, or fluid-phase uptake), or costimulatory molecule expression by DCs. However, proteasome activity in corticosterone-treated DCs or splenic DCs from stressed mice was partially suppressed, which limits formation of antigenic peptide-MHC I complexes. In addition, the lymphoid tissue-resident CD11b(-)CD24(+)CD8α(+) DC subset, which carries out cross-presentation/priming, was preferentially depleted in stressed mice. At the same time, CD11b(-)CD24(+)CD8α(-) DC precursors were increased, suggesting a block in development of CD8α(+) DCs. Therefore, glucocorticoid-induced changes in both the cellular composition of the immune system and intracellular protein degradation contribute to impaired CTL priming in stressed mice.

Distinct defects in early innate and late adaptive immune responses typify impaired fracture healing in diet-induced obesity.

Bone fractures, the most common musculoskeletal injuries, heal through three main phases: inflammatory, repair, and remodeling. Around 10% of fracture patients suffer from impaired healing that requires surgical intervention, a huge burden on the healthcare system. The rate of impaired healing increases with metabolic diseases such as obesity-associated hyperglycemia/type 2 diabetes (T2D), an increasing concern given the growing incidence of obesity/T2D. Immune cells play pivotal roles in fracture healing, and obesity/T2D is associated with defective immune-cell functions. However, there is a gap in knowledge regarding the stoichiometry of immune cells that populate the callus and how that population changes during different phases of healing. Here, we used complementary global and single-cell techniques to characterize the repertoire of immune cells in the fracture callus and to identify populations specifically enriched in the fracture callus relative to the unfractured bone or bone marrow. Our analyses identified two clear waves of immune-cell infiltration into the callus: the first wave occurs during the early inflammatory phase of fracture healing, while the second takes place during the late repair/early remodeling phase, which is consistent with previous publications. Comprehensive analysis of each wave revealed that innate immune cells were activated during the early inflammatory phase, but in later phases they returned to homeostatic numbers and activation levels. Of the innate immune cells, distinct subsets of activated dendritic cells were particularly enriched in the inflammatory healing hematoma. In contrast to innate cells, lymphocytes, including B and T cells, were enriched and activated in the callus primarily during the late repair phase. The Diet-Induced Obesity (DIO) mouse, an established model of obesity-associated hyperglycemia and insulin resistance, suffers from multiple healing defects. Our data demonstrate that DIO mice exhibit dysregulated innate immune responses during the inflammatory phase, and defects in all lymphocyte compartments during the late repair phase. Taken together, our data characterize, for the first time, immune populations that are enriched/activated in the callus during two distinct phases of fracture healing and identify defects in the healing-associated immune response in DIO mice, which will facilitate future development of immunomodulatory therapeutics for impaired fracture healing.

Different TLR signaling pathways drive pathology in experimental cerebral malaria vs. malaria-driven liver and lung pathology.

Malaria infection causes multiple organ-specific lethal pathologies, including cerebral malaria, and severe liver and lung pathologies by inducing strong inflammatory responses. Gene polymorphism studies suggest that TLR4 and TLR2 contribute to severe malaria, but the roles of these signaling molecules in malaria pathogenesis remain incompletely understood. We hypothesize that danger-associated molecular patterns produced in response to malaria activate TLR2 and TLR4 signaling and contribute to liver and lung pathologies. By using a mouse model of Plasmodium berghei NK65 infection, we show that the combined TLR2 and TLR4 signaling contributes to malaria liver and lung pathologies and mortality. Macrophages, neutrophils, natural killer cells, and T cells infiltrate to the livers and lungs of infected wild-type mice more than TLR2,4-/- mice. Additionally, endothelial barrier disruption, tissue necrosis, and hemorrhage were higher in the livers and lungs of infected wild-type mice than in those of TLR2,4-/- mice. Consistent with these results, the levels of chemokine production, chemokine receptor expression, and liver and lung pathologic markers were higher in infected wild-type mice than in TLR2,4-/- mice. In addition, the levels of HMGB1, a potent TLR2- and TLR4-activating danger-associated molecular pattern, were higher in livers and lungs of wild-type mice than TLR2,4-/- mice. Treatment with glycyrrhizin, an immunomodulatory agent known to inhibit HMGB1 activity, markedly reduced mortality in wild-type mice. These results suggest that TLR2 and TLR4 activation by HMGB1 and possibly other endogenously produced danger-associated molecular patterns contribute to malaria liver and lung injury via signaling mechanisms distinct from those involved in cerebral malaria pathogenesis.

Transcript shortening via alternative polyadenylation promotes gene expression during fracture healing.

Maturation of the 3' end of almost all eukaryotic messenger RNAs (mRNAs) requires cleavage and polyadenylation. Most mammalian mRNAs are polyadenylated at different sites within the last exon, generating alternative polyadenylation (APA) isoforms that have the same coding region but distinct 3' untranslated regions (UTRs). The 3'UTR contains motifs that regulate mRNA metabolism; thus, changing the 3'UTR length via APA can significantly affect gene expression. Endochondral ossification is a central process in bone healing, but the impact of APA on gene expression during this process is unknown. Here, we report the widespread occurrence of APA, which impacts multiple pathways that are known to participate in bone healing. Importantly, the progression of endochondral ossification involves global 3'UTR shortening, which is coupled with an increased abundance of shortened transcripts relative to other transcripts; these results highlight the role of APA in promoting gene expression during endochondral bone formation. Our mechanistic studies of transcripts that undergo APA in the fracture callus revealed an intricate regulatory network in which APA enhances the expression of the collagen, type I, alpha 1 (Col1a1) and Col1a2 genes, which encode the 2 subunits of the abundantly expressed protein collagen 1. APA exerts this effect by shortening the 3'UTRs of the Col1a1 and Col1a2 mRNAs, thus removing the binding sites of miR-29a-3p, which would otherwise strongly promote the degradation of both transcripts. Taken together, our study is the first to characterize the crucial roles of APA in regulating the 3'UTR landscape and modulating gene expression during fracture healing.