ABSTRACT
Infectious diseases propagated by arthropod vectors, such as tularemia, are commonly initiated via dermal infection of the skin. However, due to the technical difficulties in achieving accurate and reproducible dermal deposition, intradermal models are less commonly used. To overcome these limitations, we used microneedle arrays (MNAs), which are micron-scale polymeric structures, to temporarily disrupt the barrier function of the skin and deliver a bacterial inoculum directly to the dermis of an animal. MNAs increase reliability by eliminating leakage of the inoculum or blood from the injection site, thereby providing a biologically relevant model for arthropod-initiated disease. Here, we validate the use of MNAs as a means to induce intradermal infection using a murine model of tularemia initiated by Francisella novicida. We demonstrate targeted delivery of the MNA bolus to the dermal layer of the skin, which subsequently led to innate immune cell infiltration. Additionally, F. novicida-coated MNAs were used to achieve lethality in a dose-dependent manner in C57BL/6 mice. The immune profile of infected mice mirrored that of established F. novicida infection models, consisting of markedly increased serum levels of interleukin-6 and keratinocyte chemoattractant, splenic T-cell depletion, and an increase in splenic granulocytes, together confirming that MNAs can be used to reproducibly induce tularemia-like pathogenesis in mice. When MNAs were used to immunize mice using an attenuated F. novicida mutant (F. novicida ΔlpxD1), all immunized mice survived a lethal subcutaneous challenge. Thus, MNAs can be used to effectively deliver viable bacteria in vivo and provide a novel avenue to study intradermally induced microbial diseases in animal models.
INTRODUCTION
Vector-borne diseases are caused when a microbe is transmitted from a nonmammalian vector (such as a fly, mosquito, or tick) to a mammalian host, resulting in infection and illness. Transmission occurs when an infected vector takes a subsequent blood meal, typically depositing the infectious agent into the dermal layer of the skin or dermal microcirculatory system. Vector-borne diseases account for approximately 17% of all infectious diseases worldwide and are particularly prevalent in tropical and subtropical environments. More than 700,000 deaths each year result from vector-borne diseases, and poor populations are disproportionately affected (1). Furthermore, the CDC recently reported that illnesses caused by mosquitoes, fleas, and ticks have drastically increased in the United States over the past 13 years (2). The number of reported tick-borne disease cases has more than doubled, with seven new tick-transmitted infectious diseases recently being identified. This highlights the need for arthropod-transmitted disease research and relevant animal models to better understand the pathogenesis of these diseases and to develop proper treatments (2).
Researchers often use intradermal (i.d.) models of infection to study vector-borne diseases. However, this model presents unique challenges, including the fact that the needle must be inserted at a shallow angle relative to the surface of the skin, and even when the ideal angle is achieved, needle removal can result in the infectious bolus leaking back out of the penetration site or subcutaneous rather than intradermal instillation. Additionally, insertion of even a small-gauge needle results in tissue damage that may alter infection initiation and early localized responses. Finally, the use of needles presents additional exposure risks to researchers working with biosafety level 3 (BSL3) pathogens. Therefore, the development of needle-free alternatives, such as microneedle arrays (MNAs), is needed to increase research safety from pathogen exposure and the reliability and uniformity of i.d. injections (3).
MNAs are polymeric structures protruding from a planar substrate in an evenly spaced array pattern (4). Due to the length of the microneedles themselves (50 to 900 μm), MNAs that penetrate only the dermis and skin microcirculation can be designed, thereby providing the efficient transdermal delivery of a molecule or pathogen. MNAs have been most extensively characterized as improved methods for vaccine administration and drug delivery and to date have not been used as an in vivo i.d. delivery device (5–7). Donnelly et al. studied microbial penetration after microneedle puncture in vitro (using artificial membranes and excised porcine skin) and found that Staphylococcus epidermidis, Pseudomonas aeruginosa, and Candida albicans were able to transverse microneedle puncture holes (8). However, the ability of MNA-delivered microbes to induce disease in vivo has not been described. Here, we used MNAs to induce an infection using the well-characterized murine model for tularemia.
Tularemia is a febrile and potentially life-threatening illness caused by Francisella tularensis. Members of the genus Francisella are facultative intracellular Gram-negative coccobacilli, and the genus has three recognized species: Francisella tularensis, Francisella novicida, and Francisella philomiragia (9, 10). The natural reservoirs of F. tularensis are primarily small mammals, such as voles, rabbits, and other rodents. Transmission to humans can occur via direct contact with infected wildlife, via inhalation of aerosolized bacteria, and more often, through insect or arthropod vector bites (10, 11). Ulceroglandular tularemia is the most common natural disease manifestation in humans and is the result of a bite from an arthropod vector that has fed on Francisella-infected wildlife (10–12). In the United States, dog ticks, wood ticks, lone star ticks, and deer flies have all been shown to transmit tularemia to humans. From 2000 to 2010, a total of 1,208 cases of tularemia were reported to the U.S. Centers for Disease Control and Prevention. Seventy-seven percent of the reported cases occurred from May through September, a time correlated to increased human activity outdoors and peak arthropod activity (10–13).
F. tularensis subsp. tularensis has an extremely low infectious dose in humans, reported to be as few as 10 cells when inhaled; is highly virulent; and has historically been weaponized as a biowarfare agent (14, 15). As such, it is classified as a tier 1 select agent by the U.S. Centers for Disease Control and Prevention and must be handled using specific containment and biosafety measures (15). Due to the biosafety restrictions placed on virulent F. tularensis strains, F. novicida is commonly used as a surrogate in mice. F. novicida shares many important features with F. tularensis: a highly conserved genome, a comparable intracellular life cycle, and an ability to cause a rapid, tularemia-like disease in mouse infection model systems (16–18). After the delivery of F. novicida via the subcutaneous (s.c.) or intraperitoneal (i.p.) route in a mouse, the resultant tularemia-like disease is characterized by the induction of proinflammatory cytokines and dissemination via bacteremia to all organ systems. The spleen is especially affected during tularemia and becomes enlarged as a result of infiltrating inflammatory monocytes and neutrophils. The inflammatory infiltrates of the spleen lead to a shift in the ratio and relative percentages of monocytes, neutrophils, and T cells (19–23). Death ultimately results from system hypercytokinemia (21, 23). Rasmussen et al. have extensively characterized the splenic cellular response to F. tularensis subsp. holarctica LVS infection, and although the temporal dynamics of LVS infection are different from those of F. novicida infection, many of the cellular trends remain (24, 25).
Here, we used known hallmarks of tularemia pathogenesis as determinants of successful MNA-induced F. novicida infection. Our studies demonstrate that MNAs provide reproducible deposition of the bolus into the dermal layer of the mouse ear, initiating an influx of immune cells. The deposited bacteria remain viable and capable of dissemination and virulence. The observed pathogenesis and associated host response are comparable to those of previously described F. novicida murine models, including an increase in inflammatory mediators and depletion of splenic T cells. Additionally, MNAs can be used to deliver an attenuated mutant of F. novicida that protects against subsequent lethal challenge, further highlighting their use as mechanisms of vaccine delivery. As an alternative to traditional needle-based assays, MNAs increase consistency, reduce exposure to medical sharps, and reduce pain during inoculation, thereby providing an additional delivery device to study intradermal disease.
RESULTS
MNAs specifically deposit a bolus into the dermal layer, leading to immune cell infiltration.The lengths of the microneedles on the MNAs are tailored to achieve dermal penetration; in these studies, the MNAs were 650 μm from the microneedle base to the tip (4). To establish the depth of deposition, MNAs were coated with inert, green fluorescent microspheres and firmly pressed into the ear of a mouse. Using two-photon intravital microscopy, we monitored the depth of microsphere deposition in the skin. The MNA cargo was observed at depths of between −5 and −40 μm from the skin surface, suggesting that the microbeads were specifically targeted to the subepidermal layers of the ear and the dermal region (Fig. 1A; also see Fig. S1 in the supplemental material). Injury induced by a dermal lesion, such as that produced after insertion of a needle in traditional i.d. models or arthropod bites, is accompanied by the influx of innate immune cells to the site of damage (26). To determine if MNA application induces a similar cellular response, MNAs coated with fluorescent microspheres were firmly pressed onto the ear of a transgenic mouse expressing DsRed in stromal cells and green fluorescent protein (GFP) in myelomonocytic cells, including neutrophils. Resident cells were observed at the time of MNA cargo delivery (time zero), with migrating GFP-positive neutrophils being observed as early as 30 min postdelivery (p.d.) (Fig. 1B). Directed migration toward the injury site increased with time, reaching a maximum at about 2 h p.d., with this level of immune cell infiltrate persisting for more than 3 h p.d. These data suggest that MNAs induce localized innate cell responses that involve an influx of neutrophils, macrophages, and monocytes (see Video S1 in the supplemental material) similar to that previously observed after s.c. or i.p. delivery of F. novicida.
MNAs specifically deposit cargo into the dermal layer. (A) Green fluorescent microspheres (green) were introduced into the ear skin of a DsRed-transgenic mouse using MNAs. In the epidermis, keratinocytes are labeled in red. In the dermis, second-harmonic-generation imaging visualizes the fibrillar collagen network (white) interspersed with hair follicles (red). Bars = 50 μm. (B) Immune cell influx in the dermis to the site of MNA-induced injury. DsRed+/− Lyz2gfp/+ mice were used to visualize neutrophils (green) migrating between stromal cell elements (red) and collagen fibers (white) toward the MNA injury site (marked by small green fluorescent microspheres). Bars = 100 μm. (C) The percentage of viable bacteria deposited from MNAs into the dermal layer of the mouse ear was determined using variable doses of WT F. novicida, pressing the MNA against the ear and enumerating the bacteria from the ear.
MNA-induced bacterial deposition and lethality are dose dependent.To determine if MNAs can be used to deposit live bacteria, MNAs were sterilized with 70% ethanol, air dried, and incubated for 30 min in a culture of F. novicida. Excess culture was removed by blotting with a sterile cotton swab, and the MNAs were firmly pressed onto the mouse ear. Scanning electron microscopy demonstrated that sterilization with ethanol did not alter the MNA shape or integrity (see Fig. S2 in the supplemental material). To quantify the bacteria deposited from the MNAs into the animal, we performed dosing and enumeration studies using excised mouse ears. MNAs were incubated in various concentrations of F. novicida for 30 min and pressed firmly on the mouse ear for 10 s. After 15 min, the mice were euthanized; the dosed ears were aseptically removed, wiped with ethanol to remove any external bacteria, and homogenized; and the bacteria were enumerated. We calculated the resultant colonies as the number of bacteria successfully delivered into the mouse ear from the MNAs. Across the four doses tested, bacterial deposition averaged 9.4% of the initial starting concentration (1.2 × 104 CFU/ml, 10.6%; 2.6 × 104 CFU/ml, 10.3%; 1.5 × 106 CFU/ml, 10.1%; 2.5 × 106 CFU/ml, 6.4%) (Fig. 1C), suggesting a maximum dose deposition capacity and a constant relationship between the dose and the deposition rate (see Fig. S3 in the supplemental material). These rates of deposition were used to estimate the bacterial load during the subsequent animal experiments.
To determine the clinical progression of disease, two doses were administered through MNA delivery: a high dose (2.5 × 106 CFU/ml; optical density at 600 nm [OD600], 1.0) or a middose (1.5 × 106 CFU/ml; OD600, 0.7). The high dose produced 100% lethality in infected mice within 72 h postinfection (p.i.) (Fig. 2A), with mice rapidly progressing to a clinical score of 3 and higher (Fig. 2B). The middose also produced 100% lethality in infected mice; however, the time to death and clinical progression were offset by approximately 24 h compared to those seen with the high dose. This delay is suggestive of a dose dependency in terms of bacterial deposition and systemic dissemination.
Single-use MNAs induce rapid, dose-dependent lethality. MNAs incubated in an F. novicida culture at a high dose (OD600 = 1.0; n = 3) and a middose (OD600 = 0.7; n = 3) result in 100% lethality within 4 days. The lethality and clinical scores achieved with the high dose were offset from those achieved with the middose by approximately 24 h (A). All mice died within 6 h of each other (B).
Over the course of our dosing experiments, we observed disparities in lethality and clinical progression depending on the number of times that the MNAs had been used. New MNAs yielded the tightest lethality curves, with mice dying within 6 h of each other. MNAs that had been previously used once still caused lethality at both doses tested, yet the time scale was attenuated for both doses (see Fig. S4 in the supplemental material). Third-use MNAs resulted in no lethality for the middose and even further attenuated the time to death for the high dose. After use, the microneedles became visibly bent, likely resulting in less successful penetration of the dermis and the nonuniform deposition of bacteria (Fig. S4). On the basis of these results, new MNAs were used for all subsequent experiments.
Bacteria disseminate beyond the site of infection and lead to the expected pathology.The bacterial burden in blood, spleen, liver, lungs, and blood was measured to determine if MNA dosing led to bacterial dissemination and migration beyond the site of infection, as seen previously for F. novicida infection in mice. For both the high and middoses, F. novicida was detected in the spleen, lungs, liver, and blood at 24 h and 48 h postinfection (Fig. 3). A dose dependency in the bacterial burden of the spleen, liver, and blood was observed at 24 h postinfection. For both doses, the spleen showed the highest bacterial density at greater than 108 CFU/organ, followed by the liver (above 105 CFU/organ) and lung (just above the limit of detection at 102 CFU/organ). No enumerable bacteria were detected in any organs or the dosed ear from phosphate-buffered saline (PBS)-treated control mice. These data suggest that the F. novicida bacteria deposited into the dermis were viable, able to reach and enter the host circulatory system, and disseminate without being cleared by the host immune response.
The tissue response to MNA-induced i.d. infection is characterized by a high bacterial burden, tissue inflammation, and necrosis. (A, B) The bacterial burden for uninfected and infected mice was determined from the spleen, liver, lung, and blood at 24 h (A) and 48 h (B) postinfection. (C, D) The corresponding tissue pathology was observed in the spleen (C) (WP, white pulp; RP, red pulp) and liver (D). Arrows point to spots of immune cell infiltration. For all data, the mean ± SD is graphed, and significance was determined by multiple t tests. *, P < 0.02; **, P < 0.0009; ns, not significant.
The tissue response of mice to infection with F. novicida involves high bacterial burdens, as described above, leading to the formation of inflammatory infiltrate and necrotic foci (3, 27). The spleen and liver are especially susceptible to damage during infection due to the high bacterial burden. To investigate the tissue damage as a result of MNA-induced intradermal infection, the spleen and liver of infected mice were collected at 24 and 48 h postinfection (p.i.). The bacterial burdens in the spleen and liver observed for both doses at 24 and 48 h p.i. correlated with increased tissue damage. In PBS-treated mice, the architecture of the spleen remained intact at both time points, with well-defined white pulp and red pulp regions (Fig. 3C). For animals treated with the high dose, the splenic architecture was notably disrupted as early as 24 h p.i., and the spleen became increasingly inflamed and disorganized by 48 h p.i. (Fig. 3C). The red pulp and the white pulp could be more easily distinguished at 24 h p.i. in animals treated with the middose than in animals treated with the high dose (Fig. 3C). However, by 48 h p.i., the spleen was inflamed and the red and white pulp regions were no longer distinct (Fig. 3D). In the liver, foci of immune cell infiltrates were observed in the livers from all infected mice at the 48-h time point and in the high-dose group at 24 h p.i. (Fig. 3D). Immune cell infiltrate foci in the liver are a hallmark of Francisella infection described using other animal models, including animal models of F. novicida infection (28, 29).
The cytokine and cellular profiles elicited by MNA F. novicida i.d. infection are consistent with tularemia-like pathogenicity in mice.To confirm that the immunological consequences of F. novicida i.d. infection initiated using MNAs are comparable to those initiated using needle-based routes of infection, the serum levels of the cytokine interleukin-6 (IL-6) and the chemokine keratinocyte chemoattractant (KC; also called CXCL1) were analyzed. Several studies have reported that IL-6 is induced in models of Francisella infection (30–32). In our model of MNA-induced i.d. infection, the serum levels of IL-6 were significantly elevated at 24 h p.i. compared to those in PBS-treated mock-infected mice, at approximately 1,000 pg/ml in mice treated with the high dose and 900 pg/ml in mice treated with the middose (Fig. 4A). However, a dose dependency of IL-6 production was observed at 48 h p.i., with the mice treated with the high dose having more IL-6 expression than the mice treated with the middose (approximately 2,000 pg/ml and 1,400 pg/ml, respectively; P = 0.0038). Both doses led to significantly increased levels of serum IL-6 compared to those in PBS-treated mice (Fig. 4A; P = 0.0077 for mice treated with the high dose and P = 0.0004 for mice treated with the middose). We also quantified KC, a neutrophil chemoattractant previously reported in Francisella infection models (33, 34). KC serum levels in PBS-treated mice were below the limit of detection at both 24 h and 48 h after mock infection (Fig. 4B). At 24 h in treated mice, KC levels were not significantly different between high dose- and middose-infected mice (approximately 8,000 pg/ml and 7,000 pg/ml, respectively; P = 0.1012) but were significantly increased compared to those in PBS-treated mock-infected mice (Fig. 4B; P < 0.0001 for mice treated with the high dose and P = 0.002 for mice treated with the middose). A dose dependency in the serum levels of KC was observed at 48 h p.i. (approximately 26,000 pg/ml in mice treated with the high dose versus 18,000 pg/ml in mice treated with the middose; P = 0.0178). The expression profiles of both cytokines are consistent with the previously described immune responses to F. novicida infection (3, 30, 33).
MNA-initiated infection leads to T-cell depletion in the spleen and increased inflammatory cytokine levels. (A, B) The serum levels of IL-6 (A) and KC (B) were determined by ELISA. PBS-treated mice showed lower levels of IL-6 and KC than infected mice. A dose dependency in IL-6 and KC levels was observed at 48 h postinfection. (C, D) Splenocytes were evaluated by flow cytometry for the presence of immune cell subsets from mice dosed with PBS (black circles), the high F. novicida inoculum (OD600 = 1.0; red triangles), and the middle F. novicida inoculum (OD600 = 0.7; blue squares) at 24 and 48 h postinfection. (C) The numbers of Gr-1-positive granulocytes were increased in infected mice compared to the PBS-treated controls. (D) T-cell depletion was observed for both doses at both time points. *, P < 0.02; **, P < 0.01; ns, not significant.
To further confirm the character and content of the cellular immune response to F. novicida infection using the MNA intradermal route, we performed flow cytometry on the splenic immune cell populations. As expected, we observed an increase in infiltrating inflammatory monocytes and neutrophils (major histocompatibility complex class II negative CD3− CD11b+ Gr-1+) in infected animals that began at 24 h postinfection and that was sustained through 48 h postinfection (Fig. 4C). The increase in splenic monocyte and neutrophil populations was not accompanied by a similar expansion of T cells. In both the high and middose groups, we observed a decrease in the overall percentage of T-cell splenocytes at 24 h postinfection and a significant decrease in the overall percentage (P < 0.01 for both comparisons; Fig. 4D) at 48 h postinfection. Mice infected with F. novicida by MNA intradermal delivery presented a typical splenic cellular response consistent with tularemia; the infiltration of monocytes and neutrophils without the expansion of T cells led to a relative percentage shift similar to that expected with other Francisella infection models and routes (35–37).
MNAs can be used to immunize and protect mice against lethal challenge.A growing body of research has highlighted the use of MNAs as a means to provide vaccination against various diseases, including murine vaccination studies against the A/Puerto Rico/8/34 strain of influenza A virus (38). Previously, the Ernst lab has described an attenuated F. novicida mutant (F. novicida ΔlpxD1) that can be used to immunize mice against subsequent lethal challenge with wild-type (WT) F. novicida (16). LpxD acyltransferase enzymes are responsible for the temperature-regulated alterations in F. novicida lipid A biosynthesis, with LpxD1 being required for the addition of longer acyl chain (3-OH C18) at the 2 or 2′ position on the glucosamine backbone. Mice challenged s.c. with the ΔlpxD1 mutant showed no signs of illness and uniformly survived the infection. A protective immune response was also observed using an s.c. prime/boost immunization strategy. Here, we wanted to investigate if MNAs and, therefore, the intradermal delivery of the attenuated strain could likewise result in protection. Mice were immunized via MNA delivery with the F. novicida ΔlpxD1 strain (2.5 × 106 CFU/ml; OD600, 1.0) or PBS. Mice were monitored for clinical score daily, and neither group presented with any sign of illness. At 21 days postimmunization, mice were challenged subcutaneously with ∼700 CFU of WT F. novicida (∼70 times the 100% lethal dose [LD100]) and monitored for clinical score and lethality. All mice immunized with F. novicida ΔlpxD1 survived the lethal challenge, whereas all mice in the PBS control group died (Fig. 5A). Furthermore, mice immunized with F. novicida ΔlpxD1 presented no clinical signs of disease or illness after WT challenge (Fig. 5B), suggesting that a single intradermal delivery is just as effective as our previous s.c. prime/boost immunization strategy.
MNAs can be used to immunize mice against lethal challenge. MNAs incubated in F. novicida ΔlpxD1 culture (OD600 = 1.0, n = 5) or PBS (n = 5) were used to immunize mice. At 21 days postimmunization, mice were subcutaneously challenged with a lethal dose of WT F. novicida (Fn; ∼700 CFU/mouse, 70× LD50) and monitored for the clinical score (A) and lethality (B).
DISCUSSION
Microneedle arrays provide an efficient delivery mechanism to achieve dermal penetration, making it easier to study the initiation and progression of intradermal infections (4). The use of MNA-delivered F. novicida in our studies results in cellular and molecular pathologies that mirror those seen in previously reported needle-based murine models of tularemia, suggesting that MNAs can be used to model i.d. infection in mammals (9, 18). Due to the designed length of the microneedles, MNAs specifically target the dermal layer of the skin when applied to the mouse ear, allowing for a depth-specific deposition of bacteria without drawing blood (4). Importantly, i.d. infection with MNAs eliminates the need for traditional syringe needles, providing a potentially safer means to study i.d. infection when using tier 1 select agents in a BSL3 setting.
After MNA insertion, the microneedle-induced injury leads to the influx of innate immune cells, including monocytes, macrophages, and neutrophils, within minutes. The delocalized deposition of bacteria by the microneedles may also lessen the tissue damage associated with unloading a complete infectious bolus in one localized area, as is done with syringe needle-based i.d. deposition strategies (26). Our dosing experiments demonstrated a reproducible bacterial deposition rate from the MNA into the dermis (approximately 9.4% of the starting concentration). Our deposition dose of an estimated 2.5 × 106 CFU/ml (OD600 = 1.0, high dose) and 1.5 × 106 CFU/ml (OD600 = 0.7, middose) is consistent with the 100% lethal dose (LD100) in previously reported murine studies of Francisella i.d. infection models. In these studies, the dose causing a lethal i.d. infection using F. novicida or another common surrogate strain called F. tularensis LVS was in the range of 106 to 107 CFU/ml (3, 29, 39). F. novicida represents a biosafety level 2 model organism in lieu of the fully virulent biosafety level 3 F. tularensis subsp. tularensis strain. It is likely that the inoculating dose needed to achieve lethality with the fully virulent strain would be lower than what we observed using F. novicida. Our estimated deposition rate of ∼10% can serve as a guideline in future dosing and lethality studies using other strains of bacteria. Additionally, it is notable that studies investigating the amount of bacteria unloaded by an arthropod during a blood meal are limited. It has been reported that the bacterial load within a tick during or after a feeding event ranges from 104 to 108 CFU/tick (40). How this relates to the actual number of bacteria passaged from an infected tick to an uninfected mammal remains unclear, and it is likely that in vitro conditions do not accurately mirror the complexity associated with in vivo transmission between vector and mammal.
MNAs can provide an alternative means to achieve i.d. infection that could better define the dosing required to achieve dissemination and lethality. Although MNAs lose reproducibility after one use, they can be effectively manufactured as a single-use disposable device. After deposition into the dermis, F. novicida remains viable and is able to disseminate from the site of deposition. This suggests that the bacteria reach the dermal microcirculatory system and are able to migrate from the site of infection without succumbing to the host innate immune killing mechanisms. Bacterial burdens in the spleen, liver, and blood were dependent on the infectious dose at early time points, but this dependency was eliminated at later time points. Peak bacterial burdens in spleen, liver, and blood at 48 h p.i. were in the range previously reported for models of i.d. Francisella infection (3, 29, 34, 41). Additionally, F. novicida was detected in the lungs at both time points for both doses. This could indicate that mice transition to a secondary pneumonic tularemia, which has previously been described for intradermally initiated infection (3, 42). However, lung counts were obtained using whole-lung homogenates (lungs were first washed in PBS), and bronchial alveolar lavage fluid was not specifically collected. Therefore, it is possible that the observed number of CFU is from the circulating blood and is not indicative of pneumonic tularemia. The bacterial burden in the spleen and liver correlated with tissue damage. The disruption of the splenic architecture observed for both doses of MNA-induced i.d. infection is in line with previous reports and correlates with tissue inflammation and necrosis occurring partially as a result of bacterial replication (28, 29). The necrotic foci observed in the liver for both doses also support the possibility that MNAs can be used to induce tularemia in mice. The bacterial dissemination and tissue pathology that we observed using MNAs are congruent with those seen in needle-based models of tularemia.
In addition to observing a tissue pathology consistent with that seen in previously described Francisella infection models, MNA i.d. delivery also elicited cellular and molecular hallmarks similar to those seen in previously described models. Lethality in Francisella infection models is characterized by a rapid cytokine storm, including acute-phase mediators, such as IL-6, and potent chemotactic factors, such as CXCL1 (KC). Both of these cytokines were significantly induced during F. novicida MNA intradermal infection, and both play a role in the inflammatory monocyte- and neutrophil-driven pathology of the spleen (neutrophil production in bone marrow and chemoattraction for IL-6 and KC, respectively). Subsequently, we observed the cellular consequence of this potent cytokine induction. Splenomegaly, typical of Francisella infections, is characterized by infiltration of inflammatory monocytes and neutrophils, as is observed in the early phase of F. tularensis subsp. holarctica LVS infection models. We used flow cytometry to measure the cellular changes in infected mice at both doses. We also observed the expected splenic T cell depletion associated with Francisella infection models in our model. All of these results were in keeping with the expectation that lethal F. novicida infection induces a strong innate inflammatory response. Our studies suggest that the immunological profile of infected mice mirrors that of other Francisella infection models in mice, further supporting the use of MNAs as a means to initiate i.d. infection.
As with traditional syringe needle-based models, this model does not account for vector-associated factors that may contribute to infectivity and transmission in a natural setting. However, MNAs can be serially coated, as described by the Jewell lab (6). This feature may allow for the study of bacterium-protein interactions on the surface of the MNA as it is used to inoculate the host. Additionally, changes to the MNA material could be made to produce dissolvable MNAs that could be loaded with cargo (bacteria, protein, or peptide) to more efficiently deliver an infectious bolus (4). Alternative strategies could also be employed to coat the MNAs in a more sophisticated manner to more precisely control loading. For all of these reasons, MNAs offer great potential for use in vaccination. Notably, a phase I trial by Rouphael et al. recently demonstrated that dissolvable MNA skin patches can be used to effectively deliver the flu vaccine to human recipients (7). Here, we further highlight this potential using an attenuated F. novicida mutant that the Ernst lab has previously characterized as being protective (16). Mice immunized with F. novicida ΔlpxD1 were 100% protected from subcutaneous challenge with WT F. novicida, in contrast to sham-treated control mice. Our immunization studies contribute to the growing body of work demonstrating the use of MNAs in vaccination studies, specifically in studies of vaccination against a microbial disease. Beyond their use in vaccination, MNA-induced infection represents an important step in making intradermal studies more consistent and easy to perform, helping expand our understanding of dermis-initiated disease.
MATERIALS AND METHODS
Bacterial strains and growth conditions.Wild-type F. novicida strain U112 (originally obtained from Francis Nano [University of Victoria, Canada]) has been maintained as a glycerol stock at −80°C. The immunizing strain, F. novicida ΔlpxD1, was constructed as previously described and maintained as a glycerol stock at −80°C (16). For all experiments, the F. novicida strains were grown in tryptic soy broth supplemented with 0.1% cysteine (TSB-C) with aeration at 37°C and harvested during stationary growth phase.
Preparation of MNAs.Microneedle arrays (MNAs) were prepared as previously described (6). Briefly, poly(dimethylsiloxane) (PDMS) molds were prepared from a Sylgard 184 silicon elastomer kit (Dow-Corning). A total of 150 to 160 μg of poly(l-lactide) (PLLA; Sigma-Aldrich) was melted through a phase transition in the molds under vacuum (−25 in. Hg, 200°C, 40 min) and then cooled to −20°C before separating the cast PLLA microneedles from the PDMS mold. The MNAs were then cleaned and sterilized by sequential sonication in 10% bleach for 10 min and 70% ethanol for 10 min. The final arrays were washed with sterile distilled water and air dried.
Microneedle characterization.The microneedle morphology was characterized by scanning electron microscopy (SEM) using a JEOL 6700F FEG-SEM and a confocal laser scanning microscope (CLSM) using a Leica SP5X instrument. An MNA displays a total of 77 needles, each with a diameter of 250 μm at the base and a height of 650 μm.
Intravital skin imaging.Two-photon intravital imaging of ear pinnae of anesthetized mice was performed as previously described (43). Mice were anesthetized using isoflurane (Baxter, Deerfield, IL; 2% for induction and 1 to 1.5% for maintenance, with the isoflurane being vaporized in an 80:20 mixture of oxygen and air) and placed in a lateral recumbent position on a custom imaging platform such that the ventral side of the ear pinna rested on a coverslip. A strip of Durapore tape was placed lightly over the ear pinna and affixed to the imaging platform to carefully immobilize the tissue. Images were captured toward the anterior half of the ear pinna, where hair follicles are sparse. Images were acquired using an inverted LSM 510 NLO multiphoton microscope (Carl Zeiss Microimaging, Oberkochen, Germany) enclosed in a custom-built environmental chamber that was maintained at 32°C using heated air. Images were acquired using a 25×/0.8-numerical-aperture (NA) Plan-Apochromat objective (Carl Zeiss Imaging, Oberkochen, Germany) with glycerol as the immersion medium. Fluorescence excitation was provided by a Chameleon XR Ti:Sapphire laser (Coherent, Santa Clara, CA) tuned to the respective wavelengths. To measure the penetration depth of MNAs into the skin layers, the laser was tuned to 850 nm for the optimal excitation of green fluorescent microspheres (Thermo Fisher Scientific, Waltham, MA) and the generation of the collagen second harmonic signal, and a tissue depth (epidermis to dermis) of 60 μm was acquired using 1 μm z-steps. For four-dimensional data sets on innate immune cell dynamics, the laser was tuned to 930 nm for the excitation of both DsRed and enhanced GFP, and three-dimensional stacks were captured every 30 s for 4 h.
The DsRed+/+ mice and DsRed+/− Lyz2gfp/+ mice used in this study have been previously described and were on the Tyrc-2J/c-2J (B6.Albino) background to avoid the laser-induced death of light-sensitive skin melanophages (43). The mice were maintained under specific-pathogen-free conditions at an Association for Assessment and Accreditation of Laboratory Animal Care-accredited animal facility at NIAID and were used under a study protocol approved by the NIAID Animal Care and Use Committee (National Institutes of Health).
Mice and F. novicida infection.Female C57BL/6 mice, 6 to 8 weeks of age, were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in filtered cages under pathogen-free conditions and given unlimited access to sterile water and food. All animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Maryland—Baltimore and were conducted in accordance with local, state, and federal guidelines. The doses of F. novicida cultures were prepared from OD600 readings using a Beckman Coulter DU730 instrument (Beckman Coulter, Brea, CA). Dosing dilutions were made from a single culture using TSB-C until the desired OD600 was achieved (exact OD600 values within ±0.01 nm). Before use, MNAs were sterilized via submersion in 70% ethanol for 5 min and then allowed to air dry in a sterile petri dish. MNAs were submerged and incubated in the bacterial culture with gentle rocking for 30 min at room temperature. The OD600 readings of the cultures after MNA incubation did not differ from the original optical density readings. Animals were anesthetized with isoflurane, and the right ear was cleaned with an alcohol swab (Thermo Fisher Scientific, Waltham, MA). Excess medium was removed from the MNAs using a sterile cotton swab prior to infection. For intradermal (i.d.) infections, a single MNA was pressed firmly on the ear for 10 s and removed. Mice were monitored every 12 h for clinical scores and lethality (clinical scores were as follows: 0 = shiny coat, active, responsive to handling; 1 = slight lethargy, shiny coat; 2 = decreased responsiveness to handling, piloerection; 3 = decreased activity, ruffled or scruffy coat, hunched posture, rapid and shallow breathing; 4 = inactive and unresponsive, weak or ataxic; 5 = dead). Blood was collected from the lateral saphenous vein at 24 and 48 h postinfection for bacterial burden and serum cytokine analysis. At 24 or 48 h postinfection, mice were euthanized (with CO2 and by cervical dislocation), and the dosed ear, lungs, liver, and spleen were collected in accordance with the IACUC protocol (protocol number 1115004).
For immunization studies, F. novicida ΔlpxD1 was grown overnight and diluted to an optical density at 600 nm (OD600) of 1.0, as described above. MNAs were incubated in either this culture or sterile 1× PBS and pressed against the mouse ear as described above. Mice were monitored for lethality and the clinical score for 21 days after immunization. On day 21, the WT F. novicida lethal challenge dose was prepared from a 3-h subculture (1:20) of an overnight culture in TSB-C. Mice were administered 50 μl of WT F. novicida in PBS (∼14,000 CFU/ml) subcutaneously. Mice were monitored for the clinical score and lethality every 12 h after challenge for 1 week. One hundred microliters of the challenge dose and dilutions (1:10, 1:100) were plated in duplicate and incubated for 24 h. The colonies were counted to determine the exact dose (calculated to be 665 CFU/mouse). All studies were conducted in accordance with the IACUC protocol (protocol number 1115004).
Measurement of bacterial burden.Dissemination was determined by enumeration from blood, the dosed ear, lungs, liver, and one-third of the spleen. Blood was collected from the lateral saphenous vein prior to euthanasia and resuspended with citrate buffer to prevent coagulation. All organs were aseptically removed at 24 or 48 h post-MNA infection. The ears were sprayed with 70% ethanol prior to removal to eliminate viable skin-resident bacteria. The spleen and liver were weighed prior to homogenization to account for the tissue removed for histological analysis. The lungs and the dosed ear were taken whole. All tissues were homogenized in sterile PBS using a Polytron PT 1200 E instrument (Kinematica Inc., Bohemia, NY). Tenfold serial dilutions were made in PBS, and 10 μl was plated onto tryptic soy agar plates supplemented with cysteine. The resultant colonies were counted after incubation at 37°C (5% CO2) for 24 h. All dilutions and CFU spots were made in technical duplicate. Data are represented as the average for all technical and biological replicates. At least one colony from each plate was confirmed to be Francisella using a Bruker matrix-assisted laser desorption ionization protein biotyper (Bruker, Billerica, MA, USA) (44).
Splenocyte isolation and flow cytometry.Spleens were harvested and stored on ice in PBS, and single-cell suspensions were made by passing tissue through a 100-μm-pore-size nylon filter. Cells were pelleted, resuspended in ammonium-chloride-potassium (ACK) lysis buffer, incubated for 5 min at room temperature, and then washed and pelleted two times with PBS. Cells were then blocked for 20 min at room temperature with 1% bovine serum albumin, 0.1% sodium azide, PBS staining buffer. Then the cells were stained in buffer with CD3− fluorescein isothiocyanate (BD), CD11b− allophycocyanin-Cy7 (BD), and/or Gr-1–peridinin chlorophyll protein–Cy5.5 (eBioscience) for 20 min in the dark at room temperature. Following staining, the cells were washed and pelleted two times and then resuspended in PBS with 0.1% sodium azide. Data were collected using an LSR II (BD) flow cytometer, compensation among colors was performed during collection in the FACSDiva program, and data analysis was done using FlowJo (v10) software. The percentage of positive populations was calculated from a live cell population determined by size exclusion gating.
Cytokine ELISA analysis.Blood from the lateral saphenous vein was collected at 24 and 48 h post-MNA infection for all groups. Serum was isolated using Microvette tubes (Sarstedt, Nümbrecht, Germany). Serum was diluted 1:50 and analyzed using a DuoSet enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) for mouse IL-6 and KC. All reported measurements are averages for biological replicates in technical duplicate.
Tissue histology.The liver and spleen tissues were removed aseptically at 24 or 48 h after infection initiation. The upper left liver lobe was separated, and the spleen was cut into thirds under aseptic conditions. Total and partial organ weights were recorded. The liver lobe and third of the spleen were fixed in formalin overnight at 4°C. The tissues were processed and stained at the University of Maryland—Baltimore Center for Innovative Biomedical Resources Pathology and Biorepository Shared Service (fee for service [9]). Stained tissues were imaged using a Leica Aperio digital scanner (fee for service). Images were processed using ImageScope software (Leica Biosystems, Wetzlar, Germany).
Data analysis.Data are expressed as means ± standard deviations. Significance was determined using t tests in GraphPad Prism (v7.03) software. A P value of ≤0.05 was considered significant.
ACKNOWLEDGMENTS
This work was supported in part by NSF Career Award number 1351688 (to C.J.) and Alliance for Cancer Gene Therapy number 15051543 (to C.J.).
C.J. is a federal employee with the U.S. Department of Veterans Affairs.
The views in this article do not reflect the view of the U.S. Department of Veterans Affairs or the United States government.
C.J. holds an equity position in Cellth Systems, LLC, as a technical advisor.
FOOTNOTES
- Received 24 May 2018.
- Accepted 19 June 2018.
- Accepted manuscript posted online 9 July 2018.
Supplemental material for this article may be found at https://doi.org/10.1128/IAI.00406-18.
REFERENCES
- Copyright © 2018 American Society for Microbiology.
