Abstract
Infection at peripheral sites, such as the skin, activates local innate immune defenses tasked with limiting spread of the pathogen while preserving tissue integrity. T cells bearing γδ T Cell Receptors (TCR), which comprise multiple phenotypically distinct subtypes of cells, reside in normal skin, where they shape immunity to cutaneous infection, prior to onset of an adaptive immune response by conventional αβ CD4+ (TCD4+) and CD8+ (TCD8+) T cells. The mechanisms used by γδ T cells to control virus replication and tissue pathology following cutaneous infection are unknown, so we examined the role of γδ T cells in the response to cutaneous infection with vaccinia virus (VACV). Resident γδ T cells in the skin expanded and combined with recruited γδ T cells to control the pathology observed after cutaneous VACV infection. However, we observed no defect in control of local virus replication or increased systemic spread in mice lacking γδ T cells, despite induction of a cytolytic response in a specialized subset of resident γδ T cells. While examining γδ T cell-mediated control of tissue pathology following cutaneous VACV infection, we identified a unique wound healing signature associated with cutaneous virus infection that has features that are common to, but also features that antagonize, the sterile cutaneous wound healing response. Typically, tissue repair is thought to occur only after clearance of a pathogen, but the viral wound healing signature was evident prior to the peak of virus replication in the skin. Resident and recruited γδ T cells contributed to this wound healing signature through induction of multiple cytokines and growth factors required for efficient wound closure. Therefore, γδ T cells are early mediators of the wound healing response following cutaneous virus infection and are likely important in maintenance of skin barrier function and prevention of secondary bacterial infection.
Author Summary γδ T cells resident in the skin are among the first immune cell types positioned to be able to respond to a virus infection of the skin. Therefore, it was assumed that these cells in the skin play a role similar to their role after widespread infection throughout the body, namely to kill virus infected cells and slow virus replication and spread. However, we found no role for γδ T cells in control of virus replication after infection of the skin. Rather, the γδ T cells functioned as a critical component of a previously unrecognized wound healing response that is started early after virus infection of the skin, and occurs at the same time as the immune response that aims to clear the virus. This study is the first to describe both the early wound healing response after virus infection, and the role of γδ T cells in that response, and this information could allow manipulation of this response to decrease secondary bacterial infection and change scarring after virus skin infections.
Introduction
Skin resident γδ T cells are among the first dedicated effector immune cells to encounter a cutaneous virus infection, and have the potential to alter the course of the infection prior to the expansion and infiltration of conventional αβ T cells (both TCD4+ and TCD8+). Therefore, we sought to establish the role of skin resident and skin recruited γδ T cells upon control of a local cutaneous virus infection, along with their impact on systemic spread of the virus and the local pathology caused by the infection. Vaccinia virus (VACV) is an orthopoxvirus that was the most successful vaccine ever used (protecting against smallpox caused by variola virus) and now forms the basis for numerous clinical vaccine vectors. The efficacy of vaccination with VACV depends on cutaneous infection and damage [1]. There are numerous experimental and clinical indications that a cutaneous infection is the natural route of infection with VACV [2-4]. VACV is also related to both the skin-tropic orf virus, a prominent poxviral veterinary pathogen, and molluscum contagiosum, a poxvirus which infects tens of millions around the world and which can cause severe infections in immunocompromised patients [5, 6]. Thus, understanding the mechanisms and interactions between VACV and the host will provide insights about similar interactions between human and veterinary pathogens, and may identify therapeutic targets that allow manipulation of the host response, or which ameliorate pathology following infection. After systemic infection, γδ T cells exhibit cytolytic control over the replication and spread of VACV infection [7-10], but our previous work has identified key differences in how the immune system responds to control viruses after systemic infection versus the more relevant peripheral cutaneous infection. In particular, we have identified both cellular and molecular mechanisms that are induced following cutaneous VACV infection which act to control the extent of local pathology measured by lesion size and tissue loss, but which do not have large effects upon local VACV replication [11-13]. Therefore, we sought to examine the role of skin resident and skin recruited γδ T cells after cutaneous VACV infection.
Adult murine skin contains γδ T cell subsets that reside in both the epidermis and dermis and are distinguished by the Vγ TCR chain expression. Dendritic epidermal T cells (DETC) express the Vγ3 TCR chain (Garman and Raulet nomenclature [14]), whereas dermal γδ T cells express either Vγ2 or Vγ4 [15, 16]. Tissue resident cells in the skin reside among epithelial cells and are distinguished by expression of the epithelial cell adhesion molecule (Ep-CAM, CD326). In various skin pathologies γδ T cell subsets each have distinct functions, including production of IFN-γ [17-21] or IL-17A [22-25], recruitment of myeloid cells [26, 27], lysis of virus infected cells [18, 28, 29] and pro-wound healing functions [26, 30-44]. Therefore, it is likely that each resident γδ T cell type, along with recruited γδ T cells, has a differential ability to be activated, and correspondingly, a different function, following cutaneous VACV infection. In addition, γδ T cell phenotype and function are also influenced by the local microbiome [45], adding an additional wrinkle to any phenotype or function observed in uninfected or virus-infected skin.
In this study, we found that neither resident nor recruited γδ T cells are involved in the local control of VACV replication, or in the control of spread of VACV systemically, despite Vγ3 DETC displaying a cytolytic phenotype in response to cutaneous VACV infection. However, in the absence of γδ T cells, VACV infection induced a marked increase in tissue pathology over that observed in a WT situation. Neither tissue resident nor recruited γδ T cells appear to modulate any of the tissue protective functions (e.g., recruitment of myeloid cell populations, production of reactive oxygen species or Type I interferon) we have previously described as important during cutaneous VACV infection [11-13]. However, while investigating the mechanisms by which γδ T cells impact local tissue pathology we discovered induction of a unique wound healing signature induced early after VACV infection. This signature bears hallmarks of the classical wound healing response described after sterile wounding, but the viral wound healing response also displays some marked differences from the sterile response. Notably, this wound healing signature is induced early after virus infection, prior to the peak of virus replication in the skin, in contrast to the accepted paradigm, in which wound healing begins only after virus clearance [46, 47]. Our data indicate that γδ T cells mediate a requisite component of this local viral wound healing response by producing, or inducing the production of, IL-17A, IL-22, IL-10, keratinocyte growth factor (KGF) and fibroblast growth factor 9 (FGF9). Therefore, the actions of γδ T cells are likely crucial in the closure of wounded skin following cutaneous virus infection, the prevention of secondary bacterial infections within the virus induced lesions, and the maintenance of the crucial barrier function of the skin.
Results
Infected and uninfected γδ T cells in the skin are present in the foci of VACV infection and expand early after infection
VACV is a widely-used vaccine vector, and dermal administration of the vaccine most effectively induces a protective adaptive immune response [1]. However, the major complications of immunization with VACV arise either from uncontrolled virus replication, or from uncontrolled inflammation at the site of infection. The immune cells that are present in the skin at the site of infection likely play a vital role in local control of virus replication or inflammation, or both, prior to the recruitment of innate and adaptive effector cells. γδ T cells reside in the dermis and epidermis of the skin [48], and mice lacking γδ T cells are deficient in control of systemic VACV infection [7-10] and other poxviruses [49-53]. To visualize the interaction of γδ T cells and VACV within the skin, we inoculated WT mice with VACV-GFP using a bifurcated needle, a method that both generates easily identifiable foci of infection, and mimics the route of human immunization with VACV. We harvested ear tissue 4d after infection and stained tissue sections for the presence of TCRδusing antibodies. By immunofluorescence microscopy, we observed significant numbers of TCRδ+ γδ T cells that display typical dendritic morphology within the GFP+ VACV lesion, as well as some cells localized along the boundaries of the lesions (Fig. 1A). No staining for TCRδwas seen in control TCRδ-/- mice (Fig. 1B). Thus, γδ T cells are in a position to sense VACV infection, potentially lyse VACV-infected cells, and orchestrate the subsequent innate and adaptive immune response. VACV appeared to infect some γδ T cells within the infected foci. To confirm that VACV did infect γδ T cells in the skin, we infected mice intradermally with VACV, harvested ears on d4 post-infection and analyzed γδ T cells by flow cytometry. This route of infection results in a localized infection in immunocompetent mice [3, 4]. We observed that a proportion (∼11-15%) of γδ T cells in the ear expressed GFP after infection with VACV-GFP, indicating that these cells become infected with VACV (Fig. 1C).
To dissect γδ T cell responses in the skin following VACV infection, we stained single cell suspensions harvested from infected ears with antibodies to TCRδ, as well as with antibodies to conventional αβ T cells, and performed flow cytometry analysis using the gating strategy outlined in Figure 1D. As observed in a previous study [54], the numbers of γδ T cells increased early following infection (> 2-fold) and peaked at d2 post-infection (p.i.) (Fig. 1E). The expansion in number of γδ T cells appeared to represent an expansion or recruitment of all of the subsets of these cells. Notably, γδ T cells were the major lymphoid population in the VACV-infected ear until d4-5 p.i. (Fig. 1E), as NK cells are not recruited to the site of infection [11]. In addition, conventional TCD4+ and TCD8+ were recruited to the ear from the naÏve precursor pool but did not surpass numbers of γδ T cells until at least d5 post-infection (Fig. 1E). Indeed, although numerous studies have examined the TCD8+ response to VACV following infection of the skin [55, 56], these cells were in the minority compared to TCD4+ cells, which accumulate with similar kinetics and persist for much longer following infection (Fig. 1E).
To further define the γδ T cell populations in infected and uninfected skin, we quantified the numbers of Vγ3+ DETCs [14] and Vγ2+ dermal γδ T cells [15, 16] or Vγ3-Vγ2- γδ T cells. After VACV infection, staining with antibodies to Vγ3 and Vγ2 TCR chains revealed that DETC, which were consistently more abundant than other γδ T cell subsets, nearly doubled in number by d2 p.i., and then underwent a drastic contraction before recovering following resolution of infection (Fig. 1F). In contrast, Vγ2+ dermal γδ T cell numbers peaked somewhat later around d4 p.i., but underwent a slow, gradual contraction (Fig. 1F). On the other hand, the more abundant Vγ2-Vγ3- dermal γδ T cell population, which likely expresses the Vγ4 chain [16], displayed similar patterns of expansion, contraction, and recovery as DETCs (Fig. 1F). The distinct expansion and recruitment kinetics of γδ T cell populations suggests that each population may have a distinct function and role that contributes to the overall successful host response to viral infection.
A distinct γδ T cell subset displays cytolytic function after VACV infection, but does not control local virus replication or systemic spread
γδ T cells exhibit a variety of functions depending on the subtype, the stage of life at which they develop, and sub-anatomical location [57]. As γδ T cells have previously been proposed to mediate antiviral immunity via cytolytic activity [18, 28, 29], we initially examined the ability of γδ T cell subsets (Vγ3+ DETC, Vγ2+ dermal γδ T cells or Vγ3-Vγ2- γδ T cells), conventional TCD8+ or TCD4+ to degranulate in response to a broad activation signal, PMA/ionomycin treatment (Fig. 2A). Degranulation, measured via cell-surface expression of the endosomal marker CD107a in response to PMA/ionomycin, was measured on d4 and d8 post-infection, except in TCD8+, which were only present in the VACV-infected ear at d8 post-infection. We found minor cell surface staining with an anti-CD107a antibody after PMA/ionomycin treatment in TCD4+ and Vγ3+ DETC on d8 post-infection, but a marked activation-induced degranulation by the majority of Vγ3+ DETC 4 days after VACV infection (Fig. 2A). We saw only background levels of CD107a staining in the other cell types examined, either with or without of PMA/ionomycin activation. Effective cytolysis of virus-infected cells often requires the secretion of serine proteases, such as granzyme B (GzB), during degranulation [58]. Therefore, we also examined intracellular expression of GzB by Vγ3+ DETC, Vγ2+ dermal γδ T cells or Vγ3-Vγ2- γδ T cells, as well as by conventional TCD8+ or TCD4+, as above (Fig. 2B). On d8 post-infection, approximately one-third of Vγ3+ DETC, about one-half of TCD4+ and almost all of the TCD8+ expressed GzB, indicating they likely display cytolytic activity against VACV-infected cells (Fig. 2B). Earlier after infection, at d4, a similar proportion of about one-third of Vγ3+ DETC expressed GzB, and a small number of TCD4+ also had cytolytic capability (Fig. 2B). However, the majority of γδ T cells did not express GzB (Fig. 2B). Taken together, these data indicate that, of the γδ T cells present, only Vγ3+ DETC likely have the capability to directly kill VACV-infected cells in the ear.
The observation that DETC possess cytolytic activity after VACV infection drove us to examine whether γδ T cells play a direct antiviral role following cutaneous infection, as proposed previously following systemic infection [7-10]. We examined VACV titers in the ear of WT and TCRδ-/- mice, which lack γδ T cells, at d5 post-infection (Fig. 2C), a time point corresponding to the peak of virus replication [11] and at d8 post-infection (Fig. 2D), a time point before tissue loss occurs. VACV titers in the ear in TCRδ-/- versus WT mice displayed only small (∼1.5 fold) differences (Fig. 2C, D), indicating that γδ T cells do not directly or indirectly contribute significantly to the control of local cutaneous VACV replication.
A major function of the initial immune response to a peripheral virus infection is to contain the infection at the initial site, prior to recruitment of innate and adaptive effector cells which then mediate clearance of the infection. Dermal VACV infection remains primarily localized to the ear following infection [3], but removal of various immune components can allow systemic spread to the primary site of VACV replication, the ovaries [12]. However, we found no significant spread of VACV to the ovaries of TCRδ-/- mice, indicating that γδ T cells are not likely to play a critical role in restricting systemic spread of VACV (Fig. 2C, D). Therefore, γδ T cells are not required to control local VACV replication, or systemic spread of VACV.
Mice infected in the ears with VACV develop visible lesions that undergo necrosis and the necrotic tissue is then lost [3, 4]. We have previously described a role for two recruited populations of monocytes, production of reactive oxygen species and local Type I IFN production in control of the severity of pathology observed following dermal VACV infection [11-13]. However, none of the factors mentioned above are involved in control of local VACV replication or spread from the original site of infection. Therefore, we sought to examine whether γδ T cells in the skin may play a similar role in controlling the severity of pathology following VACV infection, without displaying a role in control over local VACV replication. By measuring tissue swelling, the dimensions of the lesions and ensuing tissue loss in WT and TCRδ-/- mice, we assessed the extent of tissue damage following VACV infection. TCRδ-/- mice exhibited no change in swelling of VACV-infected ear tissue in the 6 days following the initial infection when compared to that observed in WT mice (Fig. 2E). However, TCRδ-/- mice subsequently displayed visibly larger skin lesions than their VACV-infected WT counterparts on d8 post-infection (Fig. 2 F, G) and this was quantified to indicate a statistically significant change in lesion size from d6-14 post-infection (Fig. 2H). Correspondingly, there was also a visible and quantifiable acceleration and increase in severity of tissue loss in TCRδ-/- mice compared to their WT counterparts (Fig. 2 I-K). Taken together, these data indicate that γδ T cells control the severity and progression of tissue pathology at the cutaneous site of VACV infection without playing a role in the control of local VACV replication.
γδ T cell responses to VACV do not influence the local or systemic TCD8+ response to VACV
The observation that γδ T cells do not control VACV replication or spread, but do ameliorate exacerbated tissue loss following VACV infection, suggested that an overly zealous local immune host response in the absence of γδ T cells could cause an increase in local necrosis in the absence of an increase in VACV replication. Such an observation would indicate an immunoregulatory or immunosupressive role for γδ T cells. To test this we examined the effect of γδ T cells upon multiple components of the cutaneous immune response following VACV infection. TCD8+ are a vital component of antiviral immunity that are recruited to the VACV-infected ear (Fig. 1E) and localize around the periphery of the VACV lesion, thus preventing spread of the virus 4d after infection [12, 55, 59]. Therefore, we sought to examine a potential role for γδ T cells in the priming, recruitment, localization or subsequent function of TCD8+ in the anti-VACV response. We found no differences in the numbers of TCD8+ recruited to the ear either 5d (Fig. 3A) or 8d (Fig. 3B) after cutaneous VACV infection of WT or TCRδ-/- mice. We then examined the localization of TCD8+ relative to VACV-infected cells after VACV-GFP infection of WT or TCRδ-/- mice (Fig. 3C, D). In both sets of mice we observed TCD8+ around the periphery of the VACV lesion (Fig. 3C, D), indicating that there was no defect in TCD8+ localization in the absence of γδ T cells. Next, we examined the production of IFN-γ and TNFα by TCD8+ in the VACV-infected ears of both mouse strains on d5 (Fig. 3E) or d8 (Fig. 3F) post-infection. We found, on both d5 and d8 post-infection, that cytokine production by TCD8+ directly ex vivo was indistinguishable in WT vs. TCRδ-/- mice (Fig. 3E, F). These data suggest there is no overarching defect in the function of TCD8+ in the absence of γδ T cells.
To this point we have examined the recruitment, localization and function of bulk TCD8+. It was possible that the study of bulk TCD8+ of many specificities obscured subtle changes in some antigen-specific TCD8+ populations. However, it was not possible to examine the function of antigen-specific TCD8+ at the site of infection, as many cells had already been activated in the presence of VACV-infected cells (Fig.3E, F). Therefore, we examined the antigen-specific systemic TCD8+ response in the spleen 8d after dermal VACV infection of WT or TCRδ-/- mice by incubating splenocytes in the presence of 9 individual MHC Class I-binding peptides derived from VACV. We measured production of IFN-γ and TNFα, each of which can exert antiviral effects and which, when both produced by the same cells, indicate polyfunctionality that correlates with protective capacity against virus infection [60]. The proportion of TCD8+ producing IFN-γ in response to each epitope was greater than the proportion producing TNFα, but, across three replicate experiments, there were no statistically significant differences in the responses observed in WT and TCRδ-/- mice. Taken together, these results indicate that γδ T cells are unlikely to play a significant role in the priming, recruitment, localization or subsequent function of TCD8+ in the anti-VACV response. Therefore, TCD8+-mediated immunopathology is unlikely to contribute to the increased pathology we observe in VACV-infected TCRδ-/- mice.
γδ T cells do not influence the local monocyte response to VACV
It is possible that, rather than controlling immunopathology, γδ T cells coordinate the changes in the immune response that lead to resolution of inflammation and subsequent reduction of local tissue pathology. We have previously described a role for two recruited populations of monocytes in control of the severity of pathology observed following dermal VACV infection but these populations of monocytes do not have a large effect upon local VACV replication [11-13]. Both classical CD11b+Ly6C+Ly6G- monocytes and a population of CD11b+Ly6C+Ly6G+ myeloid cells are recruited to the VACV lesion following i.d. infection [11, 12, 61]. The CD11b+Ly6C+Ly6G+ myeloid cells are monocytic in nature and limit local tissue damage via production of reactive oxygen species [11]. In contrast, the classical CD11b+Ly6C+Ly6G- monocytes are attracted to the site of infection by Type I IFN-stimulated production of CCL4, and also moderate tissue damage, possibly by becoming infected and soaking up excess virions [13]. Since γδ T cells are found in the VACV lesion (Fig. 1), and are known to regulate myeloid cell activity after some insults [26, 27], we examined whether sensing of VACV by γδ T cells is required to moderate recruitment of either monocyte population. We harvested ears from infected WT or TCRδ-/- mice, and carefully gated monocyte populations to exclude innate lymphoid cells (ILCs), lymphocytes or resident or recruited dendritic cells (DC) (Fig. 4A). Five days after infection, a time point at which both virus replication and CD11b+Ly6C+Ly6G- classical monocyte infiltration peak, there was no difference in recruitment of either classical monocytes (Fig. 4B, C) or CD11b+Ly6C+Ly6G+ myeloid cells (Fig. 4B, CE) in VACV-infected WT compared to infected TCRδ-/- mice. Eight days after infection, when numbers of CD11b+Ly6C+Ly6G+ myeloid cells peak, we also observed no difference in recruitment of either classical monocytes (Fig. 4D) or CD11b+Ly6C+Ly6G+ myeloid cells (Fig. 4F) in VACV-infected WT compared to infected TCRδ-/- mice. In contrast to TCD8+, both CD11b+Ly6C+Ly6G- monocytes and CD11b+Ly6C+Ly6G+ myeloid cells enter the VACV lesion, where the CD11b+Ly6C+Ly6G- monocytes become infected, and CD11b+Ly6C+Ly6G+ myeloid cells produce ROS to moderate tissue damage [11, 59]. To examine whether γδ T cells control the localization of each myeloid cell population, we infected WT or TCRδ-/- mice with VACV-GFP and harvested on d7 post-infection, and then stained for either Ly6C or Ly6G. We observed similar staining with Ly6C and Ly6G, both inside and outside of the GFP+ VACV lesion, in both WT or TCRδ-/- mice. Therefore, our data indicate that γδ T cells do not play a requisite role in recruitment or localization of three crucial components of local cellular antiviral immunity.
IFN-γ mediates pathology in VACV-infected skin
Interferons are potent antiviral cytokines that are widely produced upon virus infection, but which are also strongly linked to the pathology of many skin conditions, including psoriasis [62] and alopecia areata [63]. We have demonstrated above that IFN-γ is produced by TCD8+ in response to cutaneous VACV infection, but that this process is not affected by the absence of γδ T cells (Fig. 3 A-H). However, direct production of IFN-γ by γδ T cells plays a role in control of multiple viral infections [17-21], including poxviruses following systemic infection [8, 50, 51, 64]. Therefore, we examined the level of mRNA encoding IFN-γ in the VACV-infected ear at various times after infection. We found a robust (∼40-fold) and reproducible induction of ifng transcript by 3 days post-infection (Fig. 5A), a time point at which we have previously described there was little to no immune cell infiltrate present [11]. The induction of ifng transcript continued to rise to ∼1-2000 fold within 7 days of infection, likely as a result of infiltration of activated TCD8+ and TCD4+ (Fig. 5A).
To examine the functional consequence of production of IFN-γ following cutaneous VACV infection, we measured the swelling of the VACV infected ear at early time points in WT vs. IFN-γR-/- mice. We found that, as early as d3 post-infection, VACV-infected IFN-γR-/- mice displayed increased swelling compared to WT mice (Fig. 5B). This correlated with development of a VACV lesion 1d earlier in VACV-infected IFN-γR-/- vs. WT mice (Fig. 5C-E) and enhanced tissue loss at later time points (Fig. 5F-H). IFN-γ is a potent antiviral cytokine that drives production of many interferon stimulated genes that act to control virus replication using a large variety of mechanisms. However, the increased local tissue pathology we observed was not a result of enhanced VACV replication in the absence of the antiviral action of IFN-γ, as WT and IFN-γR-/- mice displayed similar levels of replicating VACV in the ear at d5 post-infection (Fig. 5I). In addition, the absence of IFN-γ signaling did not alter spread of VACV from the ear, with similar low levels disseminating to spleen and ovaries by d5 post-infection (Fig. 5 J,K). Therefore, the phenotype we observe following VACV infection of IFN-γR-/- mice, namely a marked increase in tissue pathology compared to VACV-infected WT mice, without a corresponding increase in VACV replication of spread, closely mimics the phenotype we observed following VACV infection of TCRδ-/- mice.
To tease out the role of γδ T cell-mediated production of IFN-γ we examined the production of IFN-γ on d4 post-infection by staining for the intracellular cytokine, with or without activation of ex vivo isolated γδ T cell subsets by PMA/ionomycin. Somewhat surprisingly, ex vivo isolated γδ T cells of all kinds examined failed to stain for IFN-γ, even when activated with PMA-ionomycin for a number of hours before staining (Fig. 5L). As a control for our activation and staining, we examined IFN-γ production by TCD4+ and TCD8+ harvested from the lymph node of VACV infected mice on d4 post-infection, a time point prior to infiltration of these cell types to the site of infection (Fig. 5M). Although neither TCD4+ nor TCD8+ detectably produced IFN-γ in the absence of external stimuli, there was substantial and reproducible upregulation upon PMA/ionomycin stimulation (Fig. 5M), indicating that our assay was working.
Our observation that we could detect both ifng transcript (Fig. 5A) and an increase in swelling in IFN-γR-/- mice (Fig. 5B), but no detectable production of IFN-γ protein by cutaneous γδ T cells (Fig. 5J) at similar time points could potentially be explained by the relative insensitivity of the flow cytometry staining for IFN-γ protein. Therefore, we examined the levels of ifng transcript in the ears of WT vs. TCRδ-/- mice on d4 post-infection, the day on which we had previously conducted the flow cytometry analysis. As above, we found a marked induction of ifng transcript upon VACV infection of WT mice and this was enhanced slightly (∼2-fold) in the absence of γδ T cells (Fig. 5N). Therefore, γδ T cells do not contribute to IFN-γ production upon cutaneous VACV infection, a marked departure from their role following systemic infection [8, 50, 51, 64].
Because γδ T cells are not required for IFN-γ production, and leukocytes are not recruited to the infected ear until after we began to observe induction of ifng transcript, we sought to identify the cell type producing IFN-γ early after dermal VACV infection. We infected mice, harvested cells from the ear on d4 post-infection and incubated them in the presence of Brefeldin A for 6h to prevent protein secretion, stained cells with cell-surface markers to identify cell types, and then intracellularly for IFN-γ. We divided cells in the ear into EpCAM- CD45+ infiltrating immune cells, EpCAM+ CD45+ resident immune cell populations (which include resident γδ T cells) and EpCAM+ CD45- keratinocytes (KC). As in all of our flow cytometry experiments, we used fluorescence minus one (FMO) controls, in which cells were stained with all antibodies except to IFN-γ, in order to distinguish our IFN-γ signal. We found that neither EpCAM- CD45+ nor EpCAM+ CD45+ (including resident γδT) cells stained for the presence of IFN-γ on d4 post-infection (Fig. 5O, P). However, somewhat surprisingly, a small proportion of EpCAM+ CD45- KC, which comprise the majority of cells in the ear, did produce IFN-γ (Fig. 5O, P). Although the proportion of KC producing IFN-γ only increased from <0.1% of cells to ∼0.54+/- 0.07 of cells, KC outnumber all resident and recruited cells in the skin >100:1, so this increase in the number of cells is very biologically significant.
γδ T cell subsets are responsible for IL-17 production in VACV-infected skin
After we had shown that γδ T cells did not produce IFN-γ after cutaneous VACV infection our attention turned to examine the production of other cytokines that have been implicated in skin pathology. In particular, our attention turned to the γδ T cell-mediated production of IL-17A for a number of reasons. First, there is a strict division between the production of IL-17A and IFN-γ by γδ T cells [65], that may be controlled by the initiation of innate vs. adaptive signaling within these cells [21]. Second, γδ T cells are the primary producers of IL-17 outside of gut tissue [22, 23], including in cutaneous infection models. Vγ2+ dermal γδ T cells are strong producers of IL-17A following BCG infection [66] and Vγ3+ DETCs produced IL-17A after cutaneous S. aureus infection, [24, 25]. Third, γδ T cell-mediated production of IL-17A can also play a role in cutaneous pathology, driving tissue damage in psoriasis [16, 67, 68] and dermatitis [69]. Fourth, γδ T cell-mediated production of IL-17A can also play an important role in cutaneous wound healing [24]. Therefore, as γδ T cell-derived IL-17A clearly plays important roles in context-dependent anti-pathogen, as well as tissue damaging and tissue protective responses, we examined the levels of il17a mRNA transcript upon cutaneous VACV infection. There was a marked (∼10-15-fold) increase in il17a transcript in WT mice upon VACV infection (Fig. 6A). When we examined levels of IL-17A protein produced in γδTCR+ or γδTCR- cell populations by flow cytometry, using approaches similar to those outlined above, we found that >80% of the cells producing IL-17A were γδ T cells (Fig. 6B) early after infection. We further examined the ability of γδ T cell subsets (Vγ3+ DETC, Vγ2+ dermal γδ T cells or Vγ3-Vγ2- γδ T cells), to produce IL-17A directly ex vivo, or whether they possessed the ability to produce IL-17A following activation with PMA/ionomycin. A small proportion of both the Vγ2+ dermal γδ T cells and Vγ3-Vγ2- γδ T cells produced low quantities of IL-17A directly ex vivo (Fig. 6C, D), and this production was enhanced by activation with PMA-ionomycin (Fig. 6C). However, the Vγ3+ DETC, which were the cells responding to VACV infection by upregulating cytolytic activity, did not produce IL-17A (Fig. 6C, D), further indicating functional specialization of γδ T cell subsets upon dermal VACV infection. To assess the contribution of Vγ2+ and Vγ3-Vγ2- γδ T cells to the production of IL-17A following VACV infection, we measured transcript levels of il17a mRNA in uninfected or infected WT or TCRδ-/-mice, 5 days after infection. The increase in il17a transcript in WT mice infected by VACV was almost completely ablated in VACV-infected TCRδ-/- mice (Fig. 6E), indicating that γδ T cells, directly or indirectly, are responsible for almost all IL-17A production upon virus infection.
Dermal VACV infection induces a response characteristic of wound healing
Our data to this point indicated that both γδ T cells and IFN-γ can have a profound impact upon local tissue pathology following VACV infection, without a large effect upon local VACV replication or systemic spread of the virus. This phenotype is reminiscent of those we have observed in mice depleted of either of two different monocyte populations [11, 61], which lack myeloid cell production of reactive oxygen species [11], or mice lacking Type I IFN signaling [13]. Therefore, we took a step back to examine the processes induced at the site of dermal infection with VACV. We assessed the expression of various cytokines, chemokines, interferons, interferon receptors and their signaling pathways in the skin of uninfected WT mice compared to VACV-infected WT mice on d5 post-infection, a time point immediately prior to the development of lesions and subsequent tissue loss. This analysis did not directly address the role of γδ T cells, as we only examined modulation of gene expression by VACV infection in WT mice, but it did provide us with significant information with which to interrogate the role of γδ T cells (see below). In total, our gene profiling examined the expression of 160 unique transcripts 72 of which (45%) were statistically changed more than 2-fold (50 upregulated, 22 downregulated) upon VACV infection (Fig. 7A) Our focused approach allowed us to identify that ∼76-77% of the 50 upregulated and 22 downregulated transcripts had a defined role in the process of cutaneous wound healing (upregulated wound healing genes in red and downregulated wound healing genes in blue in Fig. 7A) [70-72], producing a putative gene signature for a virus induced-wound healing response in skin, shortly after infection.
Of the transcripts that were modulated by VACV infection, and have a role in cutaneous wound healing, were CC chemokines (Fig. 7B) and CXC chemokines (not shown), which function to recruit innate and adaptive immune effector cells, and all of which were upregulated. There were also marked upregulations of IL-10 superfamily members, including il10, il22 and il24 (which was upregulated ∼400-fold), and downregulation of the antagonist of the IL-10 superfamily member IL-20, il20ra (Fig. 7C). Transcripts for other cytokines with a positive role in wound healing, such as il1b, il6 and il9, were upregulated, while cytokines that have an inhibitory role in wound healing, such as il16 and il4ra, were downregulated upon VACV infection (Fig. 7D).
Growth factors such as vascular endothelial growth factor (vegf), Leukemia inhibitory factor (lif), and osteopontin (spp1) were also upregulated by VACV infection (Fig. 7E). Somewhat surprisingly, transcripts of receptors for a number of growth factors involved in wound healing, including the growth hormone (ghr), leptin (lepr) and erythropoeitin (epor) receptors, were downregulated in response to VACV infection, along with the hormones thrombopoietin (thpo) and adiponectin (adipoq) (Fig. 7A). This indicates that the presence of VACV infection in the skin may modulate the classical wound healing response, creating a unique poxvirus/wound healing signature. In addition, members of the Transforming Growth Factor-β superfamily (tgfb2, bmp2, bmp4 and bmp6) that play a role in fibrosis and scar formation in the classical wound healing response [73, 74], were also downregulated upon VACV infection (Fig. 7F), further indicating a departure from the classical response.
γδ T cells modulate expression of genes involved in wound healing after VACV infection
We expanded on our findings to examine the impact of a constitutive deficiency in γδ T cells upon the expression of cytokines and chemokines identified above as being involved in the wound healing process initiated by dermal VACV infection. As above, we utilized qPCR arrays to profile gene expression of day 5 post-infection. Importantly, in order to rule out any contribution of constitutive changes in gene expression in TCRδ-/- mice, we initially analyzed gene expression in these mice compared to WT mice, in the absence of infection. We found that a number of transcripts were upregulated in uninfected mice in the absence of γδ T cells, including cxcl13, il23a and il12b (Fig. 8A). We also found that adipoq and il16, which are genes involved in wound healing that we have previously shown to be modulated during VACV infection, were constitutively downregulated in the skin of TCRδ-/- mice, implying that a constitutive alteration in wound healing may exist in these mice (Fig. 8A). However, as we observed a change in adipoq and il16 in the absence of infection in TCRδ-/- mice, we could not draw any conclusions about the role of these genes in the response after VACV infection. We next examined changes in gene expression in uninfected vs. infected TCRδ-/- mice to reveal VACV-induced changes in gene expression in these mice. We found a similar pattern of gene expression changes to that shown in Fig. 7A, with both upregulation and downregulation of numerous cytokines and chemokines, some of which are involved in wound healing (upregulated wound healing genes in red and downregulated wound healing genes in blue in Fig. 8B). Therefore, to examine the contribution of γδ T cells to gene expression exclusively after VACV infection, we compared gene expression in WT vs TCRδ-/- mice 5 days after dermal VACV infection, a time point at which there is no significant difference in titers of VACV within the infected ear (Fig. 2D). We found that a number of transcripts are induced to higher levels in TCRδ-/- mice than WT mice, including a number of interferon-responsive chemokines such as cxcl9, cxcl13 and ccl5 (Fig. 8C). This correlates with enhanced expression of ifna2 observed in VACV-infected TCRδ-/- mice (Fig. 8B). However, we also observed that two IL-10 superfamily members, il10 and il22, which were upregulated upon dermal VACV infection (Fig. 7A), were not upregulated in VACV-infected TCRδ-/- mice (Fig. 8B), and were markedly (8-15 fold) and significantly downregulated in VACV-infected TCRδ-/- mice vs. VACV-infected WT mice (Fig. 8C). Notably, expression of neither il10 nor il22 was modulated in uninfected TCRδ-/- mice (Fig. 8A), indicating that the deficiency in il10 and il22 expression was both γδ T cell- and VACV-dependent.
To examine whether γδ T cells express IL-22 directly, or cause it’s production indirectly, we utilized Catch22 mice, in which the IL-22 promoter drives expression of the fluorescent reporter molecule, tdTomato [75]. We infected those mice i.d. with VACV expressing GFP, harvested cells on d5 post-infection and assessed tdTomato fluorescence in GFP+ and GFP- cell populations liberated from the ear. As expected, and as previously published [59] we found that a large number (∼7%) of EpCAM+CD45- KCs were VACV-infected, but we found no tdTomato fluorescence in either infected or uninfected KC populations (Fig. 8D). We next separated immune cell populations present at the site of VACV infection into resident (EpCAM+CD45+, Fig. 8E) or recruited (EpCAM-CD45+, Fig. 8G) populations. Both resident and recruited populations displayed significant levels of infection (GFP fluorescence, Fig. 8E, G), but the resident population displayed a much higher proportion of cells displaying IL-22 production than the much more numerous recruited population (tdTomato fluorescence, Fig. 8E, G). Within the resident immune cells, there were distinct populations of cells that were infected (GFP+), infected and producing IL-22 (GFP+ tdTomato+), and uninfected but also producing IL-22 (GFP- tdTomato+) (Fig. 8E). To ascertain the contribution of resident γδ T cells to IL-22 production, we also stained cells with an anti-γδTCR antibody. We were able to observe VACV infection of resident EpCAM+ γδ T cells (Fig. 8F), as previously (Fig. 1C), and a large population of IL-22-producing resident γδ T cells (Fig. 8F), but production of IL-22 by VACV-infected γδ T cells was minimal (Fig. 8F). Therefore, it is likely that other resident CD45+ cells that are VACV-infected also contribute to production of IL-22. Within the recruited (EpCAM-) immune cell populations, IL-22 production has often been attributed to a population of TCD4+, so we examined VACV infection and IL-22 production in γδTCR- lymphocyte populations (Fig. 8H) and compared this to infection of, and IL-22 production by, recruited γδ T cells (Fig. 8I). We found that infection of both populations of recruited lymphocytes was minimal (Fig. 8H, I), but that recruited γδ T cells, and not other lymphocytes, also produced IL-22. Finally, we examined the contribution of γδ T cells to il22 mRNA levels on d5 post-VACV infection of the ear, and found that, although there was a slight increase in il22 transcript levels in VACV-infected TCRδ-/- mice compared to uninfected TCRδ-/- mice, this induction was markedly and statistically significantly lower than the induction we observed in VACV-infected WT mice (Fig. 8J). Taken together, the data from Figure 8 D-J indicate that both resident and recruited γδ T cells are major producers of IL-22 during dermal VACV infection.
To examine whether γδ T cells express IL-10, the other molecule we identified as modulated in the VACV-infected ear, we utilized mice in which an internal ribosome entry site (IRES)-enhanced green fluorescent protein (eGFP) fusion protein was placed downstream of exon 5 of the interleukin 10 (Il10) gene (Vert-X mice) [76]. We were able to detect a small but distinct and reproducible population of cells liberated from the VACV-infected ear that were GFP+ 5d after infection of the IL-10-GFP reporter mice (Fig. 8L), but not of WT mice (Fig. 8K). CD45- EpCAM+ KCs did not contribute to the IL-10-GFP signal observed (not shown), and the d5 time point examined is prior to accumulation of antigen-specific TCD8+ [11, 61] that have been shown to produce IL-10 [77], so we examined IL-10 production by resident (EpCAM+) or recruited (EpCAM-) γδ T cells or myeloid cells, as previously identified in Figure 4. In contrast to the γδ T cell production of IL-22 observed above (Fig. 8F, I), we did not find IL-10 production by either resident or recruited γδ T cell populations (Fig. 8M, O). In contrast, we did find production of IL-10/GFP by resident, but not recruited, myeloid cells populations (Fig. 8N, P). Therefore, γδ T cells do not appear to express IL-10 directly upon VACV infection, but may modulate it’s expression in other ways.
Our identification of γδ T cell-modulated genes above is a minimalistic one, so we sought to examine a number of other genes that have been linked to both γδ T cells and wound healing. Two of these genes, Fibroblast growth factor 9 (fgf9, Fig. 8Q [39]) and Keratinocyte Growth Factor (fgf7, Fig. 8R [41]) were upregulated in the ear >40-fold at d5 post-VACV infection in WT mice, but fgf9 was not upregulated above the levels observed in uninfected mice in VACV-infected TCRδ-/- mice (Fig. 8Q), and the upregulation of fgf7 was markedly reduced (Fig. 8R). Therefore, γδ T cells directly or indirectly modulate the expression of multiple genes involved in the wound healing program initiated by cutaneous VACV infection.
Discussion
It is generally accepted that the local innate immune response to a peripheral virus infection is designed to slow virus replication and spread until an adaptive response can be initiated that will eliminate the virus and prevent re-infection. However, a key factor governing the extent of both the innate and adaptive immune response is that neither response should deleteriously affect the host, making it more susceptible to secondary infections, particularly via loss of barrier functions in the periphery (skin, airways etc.). Typically, this is represented in textbooks as a temporally regulated process, with the immune system gaining control over the virus, followed by a subsequent switch to reparative mechanisms that ameliorate tissue damage inflicted by both the virus and the ensuing host response. However, in this report we have described the initiation of a wound healing program in the skin concurrent with the deployment of innate antiviral strategies. The action of this wound healing program can alter tissue pathology following virus infection, independent of the control of virus replication and spread. The presence of two ongoing responses at the same time, and in very close physical proximity to each other, raises the possibility that the antiviral and wound healing responses could act synergistically to enhance each other by increasing the efficiency of monocyte recruitment, or interferon production (see below). However, it is also possible that components of one response will act to decrease the efficiency of the other. In either case, it is clear that wound healing after a cutaneous virus infection differs mechanistically from sterile wound healing, creating novel points of therapeutic intervention that could enhance wound healing and successful closure of the barrier surface to prevent secondary bacterial infection.
In a number of previous manuscripts we and others have described a role for various aspects of the local innate immune response, including two distinct monocyte populations [11, 59], reactive oxygen species [11] and Type I IFN [13], in amelioration of tissue damage following peripheral VACV infection. However, in each of these publications we found little or no effect of these components of the innate immune response upon local virus replication, despite often profound effects upon local tissue pathology [11, 13, 59]. Having outlined a wound healing program initiated upon cutaneous virus infection here, we can now retrospectively place each of these innate immune responses into that program. Recruitment of monocytes to a wound is required for effective wound healing to occur [78, 79], as is the Type I IFN receptor [80, 81]. Similarly, Ly6G+ cells are recruited to a wound [82]) and produce nitric oxide to facilitate accelerated wound healing [83]. Following VACV infection Ly6G+ cells produce ROS, which can also act to increase wound healing [84], and ablation of ROS production following VACV infection causes a large increase in pathology [11]. Therefore, all of our previous observations in which depletion or ablation of various components of the immune system has minor changes upon local virus replication, but substantial changes upon local tissue pathology, can be attributed to alterations in an early wound healing response.
Here, we initially investigated the role of γδ T cells in control of replication and spread of VACV following cutaneous infection. γδ T cells have been implicated in the host response to VACV [7-10] and other poxviruses [49-53] after systemic infection. This protective effect has been attributed to cytolytic activity against VACV-infected cells [7, 10], or to γδ T cell-mediated production of IFN-γ [8, 50]. Here, following cutaneous infection with VACV, we show that epidermal Vγ3+ DETC, which are a major population in mice but not in humans [85], acquire a GzB+ CD107a+ cytolytic phenotype. Neither the resident nor recruited dermal γδ T cell populations acquire this cytolytic phenotype. However, we and others [86] have found no difference in VACV replication in the skin of mice lacking γδ T cells, so any cytolytic contribution of these cells may be compensated for by infiltrating αβ T cells. None of the resident or recruited cutaneous γδ T cell populations appeared to be primed to make IFN-γ after VACV infection, even after in vitro restimulation, and there was a small increase in IFN-γ production in the skin of VACV-infected TCRδ-/- mice compared to WT mice. However, KCs did make IFN-γ protein shortly after VACV infection, and this contributed to control of pathology, but not to control of VACV replication.
Therefore, the functions previously attributed to γδ T cells in control of virus replication and virus-induced pathology do not play a role in the pathology observed after cutaneous infection of TCRδ-/- mice with VACV.
A sizeable portion of dermal γδ T cells produced IL-17A after cutaneous VACV infection, a cytokine that is required for efficient wound healing [24, 87]. IL-17A mRNA was reduced to almost background levels in VACV-infected TCRδ-/- mice 5 days after infection, a time point that precedes the later infiltration of TCD4+ that may produce IL-17A. Indeed, 8 days after infection, it was primarily TCD8+, not TCD4+, that produced IL-17A upon the restimulation of αβ T cells from infected skin. However, at early time points after infection, dermal γδ T cells likely moderate wound healing, at least partially, via production of IL-17A in response to cutaneous VACV infection. γδ T cells have long been known to play a role in cutaneous wound healing after activation by KCs [30-33], where they migrate to a site of injury [26, 34, 35] and produce of a number of cytokines that promote the wound healing response [36-44]. We did find a marked increase in VACV-induced pathology in the absence of γδ T cells, consistent with a role for these cells in establishment of the wound healing response following VACV infection.
Our data clearly demonstrate that γδ T cells are modulating VACV-induced skin pathology. The enhanced wound that results from VACV infection in the absence of γδ T cells prompted us to examine the range of wound healing-associated molecules induced by VACV infection that is discussed above. When we examined changes in the wound healing signature induced by cutaneous VACV infection in TCRδ-/- mice we originally anticipated that γδ T cells may alter the profile of chemokines that are induced. This is because γδ T cells have previously been reported to change recruitment of monocytes and neutrophils [26, 27], including after VACV infection [86]. In addition, IL-17A is reported to modulate neutrophil recruitment via an effect on expression of CXC chemokines [88], so in the absence of γδ T cell-produced IL-17A a defect in neutrophil recruitment would be anticipated. However, we found no discernable role for γδ T cells in modulation of chemokine or chemokine receptor expression after VACV infection, and observed equivalent recruitment of myeloid cell populations in VACV-infected WT and TCRδ-/- mice. The differences we observed from a previously published report, in which VACV-infected TCRδ-/- mice exhibited less pathology than infected WT mice, likely because of alterations in neutrophil recruitment [86], may be attributable to the cutaneous microbiome in animal facilities. We have anecdotally observed a marked difference in both the magnitude and mechanisms involved in VACV-induced pathology in mice in the presence or absence of pathogenic bacteria, so strive to ensure that mice are kept as pathogen-free as possible without rederivation into a germ-free facility. The composition of the skin microbiome has a profound effect upon wound healing [89], and future studies in germ-free animals reconstituted with different skin-resident bacteria will likely reveal roles for specific bacterial/host interactions after both “sterile” and virus-infected wounding.
When we examined the difference between the constitutive expression of wound healing-associated cytokines in WT vs TCRδ-/- mice we found two genes that were downregulated in TCRδ-/- mice, in the absence of virus infection. Expression of these two genes that encode IL-16 and adiponectin was also reduced in infected TCRδ-/- mice vs. infected WT mice. IL-16 typically increases inflammation in the skin and inhibits the wound healing response [90], but adiponectin promotes wound healing by increasing KC proliferation and migration [91]. Therefore, deficits in γδ T cells may partially account for the previously described defect in sterile wound healing [32, 44] via reduced expression of adiponectin.
Upon VACV infection of TCRδ-/- mice, we found a similar response to that observed in WT mice, with upregulation of many of the mediators of wound healing that we had observed in WT mice, and downregulation of a similar pattern of cytokines as well. However, there were two marked changes between VACV-infected TCRδ-/- and WT mice, namely a failure to upregulate expression of the IL-10 family members, il22 and il10, in TCRδ-/- mice in response to VACV infection. IL-10 is produced after VACV infection by TCD8+, but is also induced prior to the infiltration of large numbers of these cells, indicating a role for γδ T cell-mediated production. However, although γδ T cells from the liver can produce IL-10 following bacterial infection [92], we found that γδ T cells do not produce IL-10 themselves, but induce production from other cells, likely skin-resident myeloid cells such as Langerhans cells and dermal DC (Fig. 8 M, N, O). IL-10 production drives skin regeneration, likely by altering the phenotype of macrophages, as the IL-10R is not expressed by KCs [93]. Therefore, a reduction in IL-10 expression may drive part of the increase in pathology we observe following VACV infection of TCRδ-/- mice. Such an increase in pathology may actually be detrimental to the virus, as some skin-tropic poxviruses encode an IL-10 homolog that enhances wound healing [94-97], indicating that IL-10 expression may be evolutionarily beneficial for the virus during natural skin infection, perhaps by inhibiting the chances of a competing local secondary bacterial infection.
Expression of IL-22 is often associated with inflammatory skin conditions, such as psoriasis [98], where it is often co-expressed with IL-17A [99]. Expression of both IL-22 and IL-17A are most often associated with Th17 TCD4+ cells, but both cytokines are produced in large quantities in cutaneous tissue by γδ T cells [100]. Although technical issues prevented us from establishing that the same populations of γδ T cells produce both IL-17A and IL-22 following cutaneous VACV infection, it is clear that the majority of these cytokines produced at d5 post-VACV infection come from γδ T cells, and these cytokines likely drive important components of the early wound healing response. IL-22 acts on dermal fibroblasts to drive expression of extracellular matrix proteins [101], and on KCs to increase proliferation [98, 102, 103] via miR-197-driven mechanisms [104], as observed after bacterial skin infection [105]. In addition to the IL-10 family members, we also found that expression of both fgf7 (encoding KC Growth factor) and fgf9 were markedly reduced in VACV-infected TCRδ-/- mice, consistent with reports of their production by γδ T cells and roles in cutaneous wound healing [39, 44]. Therefore, γδ T cells contribute in multiple ways to the cutaneous wound healing response following VACV infection.
In summary, we find here that neither resident nor recruited γδ T cells, nor cytokines produced by these cells, are involved in control of virus replication or spread following cutaneous infection. Rather, we describe here a uniquely configured wound healing response initiated in the skin of virus-infected mice prior to the peak of virus replication and before adaptive immune mechanisms have been deployed successfully to clear the infection. We find that both resident and recruited γδ T cells are part of this induced wound healing response via production of IL-17A, IL-22 and induction of IL-10 in other cells, and that a deficit in γδ T cells causes a profound increase in tissue pathology following infection. These findings are important in understanding how wound healing is mediated following a cutaneous virus infection in comparison to the paradigm of sterile wound healing. A prompt and appropriately controlled wound healing response is crucial to prevent secondary bacterial infections that could be deleterious for the virus-infected host, but also potentially for the virus itself.
Materials and Methods
Mice
C57BL/6 (wild-type, WT) mice were purchased from Charles River Laboratories or Jackson Laboratories. Breeding pairs of B6.129P2-Tcrdtm1Mom/J (TCRδ-/-) [106], IL-10/GFP (Vert-X) reporter mice [76], and IL-22/tdTomato (Catch22) reporter mice [75] were purchased from Jackson Laboratories. These mice were on a WT background after a minimum of 12 backcrosses to C57BL/6 and bred in the specific-pathogen-free animal facility at the Penn State Hershey College of Medicine. All animals were housed and cared for according to guidelines from the National Institutes of Health and American Association of Laboratory Animal Care (AALAC). The Penn State Hershey College of Medicine Institutional Animal Care and Use Committee (IACUC) approved all animal experiments and procedures.
Viruses and infections
Stocks of VACV strain WR were produced in 143B TK- cells and purified from cell lysate following ultracentrifugation through a cushion of 45% sucrose. VACV-GFP was previously described [107]. For intradermal (i.d.) infections, mice aged 7-10 weeks were anesthetized with ketamine/xylazine and injected with 104 PFU of VACV in <10 μL in each ear pinna.
To monitor pathogenesis in the ears, ear thickness was measured using a 0.0001 in. micrometer (Mitutoyo, Aurora, IL). Lesion progression and subsequent tissue loss were subsequently measured daily. To measure titers of virus in vivo, ears and ovaries (the target organ of VACV systemic spread [12]) were freeze-thawed three times, homogenized, and sonicated, then titers in cell lysates were assayed by plaque assay on 143B TK- cells as previously described [61]. Plaques were counted two days later.
Cell isolation and flow cytometry
Pairs of ears from each mouse were split into dorsal and ventral halves, minced, and digested in a solution of 1 mg/mL collagenase type XI (Sigma-Aldrich, St. Louis, MO) in media supplemented with 2% FBS and 5 mM CaCl2 for 1 hour at 37°C, 5% CO2. Collagenase was quenched with media containing 5% FBS and 5 mM EDTA. Digested tissue was passed through 40 μm nylon cell strainers to create a single cell suspension. For intracellular cytokine staining and CD107a degranulation assays, cells from 3 pairs of ears were pooled and 104 of those cells stimulated prior to staining for flow cytometry (see below).
Blockade of FcR-mediated binding of mAbs and subsequent staining of cells was performed in supernatant from flasks of 2.4G2 hybridoma cells supplemented with 10% normal mouse serum. All mAbs used were purchased from BD Pharmingen: CD45 (30-F11), CD3ε (145-2C11), TCRδ(GL3), CD4 (RM4-5), NK1.1 (PK136), CD19 (1D3), CD90.2 (53-2.1), CD11b (M1/70), Ly6C (AL-21), Ly6G (1A8), CD107a (1D4B), Granzyme B (GB11), IL-17A (TC11-18H10), TNFα (MP6-XT22) and IFN-γ (XMG1.2). We also utilized the following from Biolegend: Vγ2 TCR (UC3-10A6), Vγ3 TCR (536) and CD64 (X54-5/7.1). CD8α (53-6.7) and CD11c (N418) were obtained from eBioscience. In addition, PE-Cy7 conjugated streptavidin (eBioscience) was used to label biotin-conjugated antibodies. To stain for granzyme B, cells were stained for surface markers, fixed in 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), then permeabilized and stained intracellularly for granzyme B in 2.4G2 supernatant containing 10% normal mouse serum and 0.5% saponin (Sigma). Sample data was acquired on either an LSR II or LSR Fortessa flow cytometer (both from BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Tree Star, Ashland OR).
Intracellular cytokine staining assay
Single cell suspensions of ears were stimulated for 5 hours at 37°C, 5% CO2 with 50 ng/mL phorbol myristate acetate (PMA; Sigma) and 1 μg/mL ionomycin (Sigma) or unstimulated in the presence of 10 μg/mL brefeldin A (BFA; Sigma). For T cell stimulations, lymphocytes were isolated by centrifugation over Lymphocyte Separation Medium (Cambrex) and then stimulated for 4 h with 1 μM VACV peptide prior to the addition of BFA. VACV-derived peptides B8R, A8R, A3L, A23R, K3L, A47L, A42R, A19L, 10G2 have been previously described [108]. Following PMA/ionomycin or peptide stimulation, cells were blocked in 2.4G2 supernatant containing 10% mouse normal mouse serum and then stained for CD45, CD3ε, TCR δ, Vγ2 TCR, Vγ3 TCR, and CD4. Cells were fixed in 2% paraformaldehyde then permeabilized and stained for intracellular IL-17A and IFN-γ in 2.4G2 supernatant supplemented with 10% normal mouse serum and 0.5% saponin. Net frequencies and numbers of cytokine-positive TCD8+ were calculated by subtracting the unstimulated background response.
CD107a degranulation assay
Cells from ears were stimulated for 5 hours at 37°C, 5% CO2 with 50 ng/mL PMA and 1 μg/mL ionomycin or unstimulated in the presence of 1.5 μg/mL monensin (Sigma) and PE-conjugated rat anti-mouse CD107a. Following stimulation, cells were blocked in 2.4G2 supernatant containing 10% normal mouse serum and then stained for CD45, CD3ε, TCR δ, Vγ2 TCR, Vγ3 TCR, and CD8α.
Immunofluorescence Microscopy
Ears were harvested and embedded in Tissue-Tek OCT (Sakura Finetek), then rapidly frozen by immersion in liquid nitrogen-cooled 2-methyl butane, and kept at -80 °C overnight. Cryostat sections (10-12 μm) were cut at -20°C, mounted on glass slides, air-dried for 2-3 hours, fixed for 10-15 minutes in 1% paraformaldehyde (pH 7.4), air-dried again for 30 minutes, and stained with antibodies to TCRδ, Ly6C, Ly6G or CD8 (clones as above). Positive signal was revealed by subsequent staining with fluorescently-labeled secondary antibodies. Staining was visualized using an Olympus 1×81 deconvolution microscope and Slidebook 5.0 digital microscope.
Quantitative PCR
Tissues were harvested, digested as above, and total RNAs were extracted using RNeasy Plus Mini Kit (Qiagen) with DNase treatment according to the manufacturer’s protocol. For qPCR using Taqman Gene Expression Assays (Applied Biosystems) or Universal Probe Library (Roche), cDNA was prepared using the Hi-Capacity cDNA Synthesis Kit (Applied Biosystems). For qPCR using RT4 PCR Profiler Arrays (Qiagen), cDNA was prepared using RT4 First Strand Kit (Qiagen). qPCR was carried out on a StepOnePlus (Applied Biosystems) with either FastStart Universal Probe Master Mix (Roche) or RT4 SYBR Green qPCR Master Mix (Qiagen). For Taqman and FastStart Universal Probe assays, changes in gene expression are expressed as fold change using the ΔΔCt calculation method against naÏve mice of the same genotype with gapdh as the housekeeping gene. For RT4 PCR Profiler Array data, changes in gene expression are displayed as mean fold change between groups of mice relative to a panel of “housekeeping” genes. SYBR Green primers were as follows: fgf7 forward 5′-ATAGAAACAGGTCGTGACAAGG-3′ reverse 5′-CAGACAGCAGACACGGAAC-3′ fgf9 forward 5′-GTAGAGTCCACTGTCCACAC-3′ reverse 5 ′-CAACGGTACTATCCAGGGAAC-3′. Taqman primer/probe sets (Thermo Fisher) were as follows: il17a (Mm00439618_m1), il22 (Mm00444241_m1), gapdh (Mm99999915_gl) and ifng (Mm01168134_m1).
Author Contributions
Conceived and designed the experiments: IER, EL, AMN and CCN. Performed experiments: CCN, EL, IER, TEK, and NJP. Analyzed the data: EL, IER, TEK, and NJP. Wrote the paper: CCN.
Acknowledgements
We thank colleagues Drs. Jon Yewdell and Jack Bennink for the WR VACV used in these studies. We thank Melanie Epler and Jennifer Mellinger for excellent technical support of these studies. We also the Penn State College of Medicine flow cytometry core facility and Karen Briar, Robin Goshorn, Jeanette Mohl, Dr. Tim Cooper, and Dr. Tiffany Whitcomb for essential animal handling and veterinary assistance. We also thank Dr. Nick Buchkovich for critical review of the manuscript.
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