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.

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.

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.

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.

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.