The differential in vivo contribution of spinal α2A- and α2C-adrenoceptors in tonic and acute evoked nociception in the rat

Spinal α2-adrenoceptor induces analgesia by neuronal inhibition of primary afferent fibers. This family receptor coupled to G i/o proteins can be subdivided into three functional subtypes: α2A, α2B, and α2C-adrenoceptors, and current evidence on spinal analgesia supports the relevance of α2A and seems to exclude the role of α2B, but the functional contribution of α2C-adrenoceptors remains elusive. The present study was designed to pharmacologically dissect the contribution of spinal α2-adrenoceptor subtypes modulating tonic or acute peripheral nociception. Using male Wistar rats, we analyzed the effect of spinal clonidine (a non-selective α2A/α2B/α2C-adrenoceptor agonist) and/or selective subtype α2-adrenoceptor antagonists on: 1) tonic nociception induced by subcutaneous formalin (flinching behavior) or 2) acute nociception induced by peripheral electrical stimulus in in vivo extracellular recordings of spinal dorsal horn second-order wide dynamic range (WDR) neurons. Clonidine inhibited the nocifensive behavior induced by formalin, an effect blocked by BRL 44408 (α2A-adrenoceptor antagonist) but not by imiloxan (α2B-adrenoceptor antagonist) or JP 1302 (α2C-adrenoceptor antagonist). Similarly, spinal BRL 44408 reversed the clonidine-induced inhibition of nociceptive WDR activity. Interestingly, spinal JP 1302 per se produced behavioral antinociception (an effect blocked by bicuculline, a preferent GABAA channel blocker), but no correlation was found with the electrophysiological experiments. These data imply that, at the spinal level, 1) presynaptic α2A-adrenoceptor activation produces antinociception during acute or tonic nociceptive stimuli; and 2) under tonic nociceptive (inflammatory) input, spinal α2C-adrenoceptors are pronociceptive, probably by the inactivation of GABAergic transmission. This result supports a differential role of α2A and α2C-adrenoceptors modulating nociception.

Introduction
At the spinal dorsal horn level, α 2 -adrenoceptor activation has been related to the inhibition of neural transmission and analgesic actions (Pertovaara, 2006;Bahari and Meftahi, 2019). From a mechanistic perspective, activation of this receptor which is canonically coupled to G i/o proteins, inhibits adenylyl cyclase, inactivates Ca 2+ channels, and enhances inwardly rectifying K + channel activity, leading to neuronal hyperpolarization with a diminution of neural transmission (Pan et al., 2008). In vivo electrophysiological recordings of second-order wide dynamic range (WDR) cells in the spinal dorsal horn have shown that spinal clonidine (α 2adrenoceptor agonist) selectively inhibits the neuronal activity of nociceptive primary afferent fibers . As reviewed by Pertovaara (2013), agonists to this receptor consistently produce antinociception in behavioral and electrophysiological experiments. Indeed, intrathecal clonidine has been successfully used in humans as a potent analgesic (Eisenach et al., 1996;Yaksh et al., 2017;Schwartz et al., 2022).
However, we must keep in mind that α 2 -adrenoceptors have been divided into three functional subtypes, namely α 2A , α 2B , and α 2C (Bylund et al., 1994), and researchers have attempted to dissect how these receptor subtypes contribute to spinal pain transmission. Briefly, molecular assays suggest that the three receptor subtypes are expressed in the spinal cord and dorsal root ganglion cells (Aoki et al., 1994;Stone et al., 1998;Nicholson et al., 2005), and current functional evidence supports the notion that α 2A -adrenoceptor activation plays a key role in spinal antinociception, whereas α 2Badrenoceptors seem not to contribute to spinal antinociception (for refs., see Pertovaara, 2006;Pertovaara, 2013). In contrast, in the case of α 2C -adrenoceptors, although Fairbanks et al. (2002) suggest that spinal activation of this subtype receptor plays an antinociceptive role, the evidence offered by Malmberg et al. (2001) refuses these data showing that this receptor does not play any role in nociception. In both cases, the experiments were performed using transgenic mice and non-selective ligands. Hence, the function of this subtype receptor remains obscure.
Most importantly, although current literature supports the notion that the main effect of α 2 -adrenoceptors is inhibition of nociceptive transmission, some in vitro/in vivo experiments suggest that noradrenergic transmission at the spinal cord level could also be pronociceptive (Hantman and Perl, 2005;Chen et al., 2008;Gassner et al., 2009;Hickey et al., 2014;Kucharczyk et al., 2022). For example, using spinal cord slices and electrophysiological recordings (patch-clamp) at lamina II/III, it has been shown that some GABAergic neurons can be hyperpolarized by noradrenaline via α 2 -adrenoceptor activation (Gassner et al., 2009); in this study, the subtype receptor was not analyzed. Furthermore, also in spinal cord slices, but using immunohistochemistry, Chen et al. (2008) showed that α 2Cadrenoceptor activation inhibits the veratridine-induced opioid release. These in vitro data may imply that α 2Cadrenoceptors may play a pronociceptive role. In any case, the functional role of α 2C -adrenoceptors modulating spinal nociception has been hampered (contradictory) in part by the lack of selective ligands used.
On this basis, the present study aimed to functionally dissect α 2A/2B/2C -adrenoceptors in spinal nociception. Here, we analyzed the effect of spinal clonidine (a non-selective α 2A/ α 2B/ α 2C -adrenoceptor agonist) and/or selective subtype α 2A/ α 2B/ α 2C -adrenoceptor antagonists (see Table 1) on: 1) tonic nociception (flinching behavior) induced by subcutaneous formalin or 2) acute nociception induced by peripheral electrical stimulus in in vivo extracellular recordings of spinal dorsal horn second-order wide dynamic range (WDR) neurons. The data showed that the presynaptic α 2A -adrenoceptor produces robust antinociception during acute or tonic nociceptive stimuli. In contrast, under a tonic nociceptive stimulus, α 2Cadrenoceptors seem to induce nociception by inactivating GABAergic transmission.

Experimental animals and ethical standards
A total of 115 male Wistar rats (295 ± 15 g) from the Neurobiology Institute Animal House were used in the present experiments. Rats were divided into two main sets (80 rats for behavioral tests and 35 rats for electrophysiological recordings; see Figure 1). The rodents were housed in the satellite bioterium of our laboratory on a 12:12 h light and dark cycle (lights on at 07:00 h) at constant temperature (22°C ± 2°C) and humidity (50%) with food and water ad libitum. All experimental procedures were performed during the light phase of the cycle (10:00-19:00 h). Furthermore, all animal protocols in this investigation were approved by our Institutional Ethics Committee, following the Guide for the Care and Use of Laboratory Animals in the United States (NIH publication 86-23), IASP ethical guidelines (Zimmermann, 1983), and ARRIVE guidelines for reporting experiments involving animals (McGrath et al., 2010). At the end of the experiments, the animals were halted in a CO 2 chamber (formalin test) or by an overdose of pentobarbital (electrophysiological experiments).

Surgical procedures for behavioral experiments (intrathecal surgery)
Chronic catheterization of the intrathecal (i.t.) subarachnoid space was performed as described by Yaksh and Rudy (1976).
The rats were anesthetized with a ketamine-xylazine (75-10 mg/kg, i.p.) and placed in a stereotaxic frame (Kopf Instruments, United States) with the dorsal part of the head and neck previously shaved. The atlanto-occipital membrane was exposed and pierced, and a polyethylene catheter (PE-10, 9.0 cm length) was inserted intrathecally and advanced caudally to the All data are given as pK i . a (Jasper et al., 1998) or pK D . b (Proudman et al., 2022) values at human receptors.

FIGURE 1
Experimental protocols showing the number of animals used in the present study. The animals were divided into two main sets to perform behavioral or in vivo electrophysiological recordings.
Frontiers in Pharmacology frontiersin.org level of the thoracolumbar junction. The wound was then sutured, and the animals were housed in individual cages to recover from surgery for 5 days before the formalin test.

Formalin-induced tonic nociception and study design
The tonic nociception experiments using the 1% formalin test (Dubuisson and Dennis, 1977;Wheeler-Aceto and Cowan, 1991) were performed by the same tester, blinded to the pharmacological treatment. Rats were placed in open Plexiglass ® observation chambers for 30 min for three consecutive days to allow them to become familiar with their surroundings. On the third day, and after 30 min in the Plexiglass ® chamber, they were removed for formalin administration. Fifty microliters of diluted formalin (1%) were subcutaneously (s.c.) injected into the dorsal surface of the right hind paw with a 30-gauge needle. The animals were then returned to the chambers, and nocifensive behavior was observed immediately after the formalin injection. Nocifensive behavior was quantified as the number of flinches of the injected paws during 1 min periods every 5 min for up to 1 h after injection. Flinching was characterized as a rapid and brief withdrawal or as a flexing of the injected paw. As previously reported, formalin-induced flinching behavior was biphasic. The initial nociceptive phase (0-10 min) was followed by a prolonged, persistent response (15-60 min). For i.t., administration, the α 2A/2B/2C -adrenoceptor agonist (clonidine) or selective antagonist (BRL 44408 to α 2A -, imiloxan to α 2B -, or JP 1302 to α 2C -adrenoceptors) were given in a volume of 10 μl using a Hamilton ® syringe.
Since JP 1302 alone inhibited the formalin-induced nociception, the involvement of GABAergic mechanisms was suspected (see discussion section). To test this hypothesis, 0.3 nmol bicuculline was given 10 min before JP 1302 (n = 5 rats; 1 nmol). Also, the per se effect of bicuculline on formalin flinches was assessed (n = 5 rats).
Doses and drug administration schedules for clonidine, BRL 44408, and imiloxan were selected based on previous reports (O'Neill and Haigler, 1985;Young et al., 1989;Omote et al., 1991;Uhlén et al., 1994;Erne-Brand et al., 1999;Dwyer et al., 2010) and pilot experiments in our laboratory. Behavioral or motor function changes induced by the different treatments were assessed in catheterized rats from all groups by testing the animals' ability to stand and walk in a normal posture, as proposed elsewhere (Chen and Pan, 2001).

Surgical procedures for electrophysiological experiments
Animals were anesthetized with urethane (2 g/kg), and then an intratracheal cannula was inserted for artificial ventilation (65-75 strokes/min; model Ro Vent ® , Kent Scientific Corp. United States). Subsequently, animals were mounted onto a stereotaxic frame (Kopf Instruments, United States) and secured in a spinal cord unit frame. The lumbar vertebrae were fixed to improve stability at the recording site to perform a laminectomy to expose the L2-L4 spinal cord segments. The dura was carefully removed, and the exposed spinal cord was covered with isotonic saline to avoid desiccation. The animals were not paralyzed, and we did not observe any withdrawal response during peripheral electrical stimulation. End-tidal CO 2 was monitored with a Capstar-100 End-tidal CO 2 analyzer (CWE Inc., United States) and kept between 3.0% and 3.5% by adjusting the stroke volume. Core body temperature was maintained at 38°C using a circulating water pad.

Extracellular unitary recordings and study design
Extracellular unitary recordings were made using seven quartz-Pt/W microelectrodes (impedance 4-7 MΩ) mounted in a multichannel microdrive with an integrated preamplifier. This multi-electrode system was manipulated with the 7-channel version of the fiber-electrode manipulator "System Eckhorn" using Eckhorn Matrix multiuser software (Thomas Recording GmbH, Germany). The microelectrodes were lowered (400-900 μm from the surface) in small steps (2-5 μm/s) into the superficial laminae of the left dorsal horn segments to search for single-unit discharges. For each recorded cell, the specific somatic receptive field (RF) was located by gently tapping on the entire ipsilateral glabrous surface of the hind paw. When we found a RF, electrical stimulation using an S88 stimulator (Grass Instruments Co., United States) was then applied. In this case, two needles (27 G) attached to a stimulus isolator unit (PSIU6 model, Grass Instruments Co., United States) were subcutaneously inserted into the RF of the recorded neuron. The electrical stimulation was then conducted and consisted of 20 stimuli at 0.2 Hz with a 1 ms pulse duration at 1.5 times the threshold intensity (0.1-3 mA) required to evoke a C-fiber discharge. It is interesting to note that using this protocol, the spikes associated with activation of Aβ-, Aδ-, C-fibers, and post-discharge can be observed, but the wind-up was not consistently elicited.
The extracellular neuronal activity induced by electrical stimulation of the RF was amplified ×100 (1700 Differential AC amplifier, A-M Systems, United States), digitalized, and discriminated using CED hardware and Spike 2 software (v5.15; Cambridge Electronic Design, United Kingdom). Raw and discriminated signals were fed through an audio monitor (model 3300, A-M Systems, United States) and displayed on an oscilloscope (TBS1064, Tektronix Inc., United States). Waveforms and recorded spike trains were stored on a hard drive for offline analysis. Evoked activities of the spinal dorsal horn WDR neurons were recorded and analyzed as cumulative frequency and peri-stimulus time histograms (PSTH) to detect the occurrence of neuronal responses. On this basis, the stimulating threshold to evoke action potentials and their frequency of occurrence, resulting from the stimulation of the peripheral RF on the hind paw, were attributed to the recruitment of Aβ-, Aδ-, and C-fibers. Considering the distance between the RF and the recording electrode, the peak latencies observed correspond to peripheral conduction velocities within the Aβ-, (0-20 ms), Aδ-(21-90 ms), C-fibers (90-350 ms), and post-discharge (350-800 ms) (Urch & Dickenson, 2003;González-Hernández et al., 2019;Gamal-Eltrabily et al., 2020). By using this protocol, we exclusively recorded WDR cells, a type of second-order neurons receiving input concomitantly from non-nociceptive (Aβ-type) and nociceptive (Aδ-and C-type) fibers; in addition, postdischarge was also analyzed.
Indeed, during the search of WDR cells responding to peripheral tactile and electrical RF stimulation, some neurons can be classified as only tactile sensitive and, in minor proportion, nociceptive specific; but these types of cells were not further analyzed. Thus, the number of action potentials in response to 20 RF stimuli was compared before (basal, t = 0) and after treatments. In these experiments, a control group without treatment was used (n = 5).
Accordingly, the neuronal evoked responses were evaluated immediately after (basal; t = 0) clonidine (1 or 10 nmol; n = 5 cells, each) or vehicle (isotonic saline, 0.9% NaCl; n = 5 cells) administration and at 10, 20, 30, 40, 50, and 60-min posttreatment. Since in the formalin test, clonidine-induced flinching inhibition was reversed by BRL 44408 but not by imiloxan or JP 1302, the role of the α 2A -adrenoceptor was evaluated by spinal administration of BRL 44408 (1 nmol; n = 5 cells; given 10 min prior clonidine). Furthermore, considering that JP 1302 exhibited antinociceptive effects in the formalin test, we tested the effects of this α 2C -adrenoceptor antagonist (1 or 10 nmol; n = 5 cells, each) on the electrophysiological responses of WDR cells. The vehicle and compounds were given at the spinal cord level (topical) in a total volume of 10 μl using a Hamilton ® syringe.

Data presentation and statistical analysis
The data in the figures are presented as mean ± S.E.M., (standard error of the mean). In all cases and before performing a parametric statistical analysis, we checked for normality using the Shapiro-Wilk test (p > 0.05). In the formalin test, curves were constructed by plotting the number of flinches as a function of time. The area under the number of flinches against time curves (AUC), an expression of the duration and intensity of the effect, was calculated by the trapezoidal rule, and a one-way analysis of variance (ANOVA) was performed.
For electrophysiological experiments, a baseline neuronal response was established after an identified neuron had a ≤10% variation in the neuronal responses induced by RF stimulation during five consecutive tests (5 min between each trial). The number of basal (t = 0) evoked potential (total spikes and number of Aβ-, Aδ-, C-fibers and post-discharge) in the different experimental groups were analyzed; since the normality test failed, a Kruskal-Wallis one-way analysis of variance (ANOVA) on ranks was performed (see Table 2). On this basis, and to normalize the data, the evoked potentials induced by electrical stimulation of the paw were expressed as a percentage change from the respective baseline. Thus, the baseline value refers to the evoked neuronal response before spinal treatment with clonidine or antagonists. To evaluate the stability of the recorded neurons (only for the control group and vehicle group) across the 60-min time frame, we used a one-way repeated measures ANOVA. The difference in neuronal activity evoked within one group of animals before and after treatments was compared using a two-way repeated-measures ANOVA. In addition, the temporal course was adjusted to obtain global neuronal activity due to the treatment (box and whisker plots); in this case, a one-way ANOVA was performed.
Frontiers in Pharmacology frontiersin.org The ordinary one-way ANOVA was followed (if applicable) by the Newman-Keuls post-hoc test, whereas in the case of the two-way repeated-measures ANOVA, Holm-Sidak's multiple comparison test was followed. Furthermore, sphericity was not assumed in the case of repeated measures ANOVAs, and corrections to degrees of freedom were made according to the Greenhouse-Geisser method. Differences were considered statistically significant when p < 0.05. Graphs and statistical analysis were done using GraphPad Prism V6.0 software (United States). Complete statistical analysis is detailed in Tables 2, 3.

Intrathecal clonidine inhibits flinching behavior induced by formalin
Subcutaneous (s.c.) formalin injection into the right hind paw produced a typical flinching behavior characterized by a biphasic time course (Figure 2A; control curve). Phase I of the nociceptive response began immediately after formalin injection and gradually declined (≈10 min). Then, phase II started about 15 min after formalin injection and lasted 1 h. I.t administration of clonidine (0.1, 1, and 10 nmol) inhibited the formalin-induced flinching behavior during phases I and II ( 3.2 Effect of α 2A -(BRL 44408), α 2B -(imiloxan), and α 2C -(JP 1302) adrenoceptor antagonists in the clonidine-induced behavioral antinociception As shown in Figure 3A, the antinociception induced by 1 nmol clonidine was attenuated by 1 nmol BRL 44408 and remained unaffected by 0.1 nmol BRL 44408, 10 nmol imiloxan, or 1 nmol JP 1302. In particular, 1 nmol BRL 44408 partially reverted the clonidine effect during phase I ( Figure  3B) and abolished the clonidine-induced antinociception during phase II ( Figure 3C).
When we tested the per se effects induced by the antagonists ( Figure 3D), we found that i.t. administration of vehicle (0.9% NaCl solution, 10 µl), BRL 44408 (0.1, 1 nmol) or imiloxan (10 nmol) did not have a statistical difference (see Table 2 for details) on the flinching behavior induced by formalin; in contrast, i.t. JP 1302 significantly reduced the number of flinches during phase I and II ( Figures 3E,F).
Since the behavioral experiments showed that clonidine inhibits the formalin-induced nociception via α 2Aadrenoceptor activation, a set of electrophysiological recordings of the second-order WDR dorsal horn spinal cord cells were performed to correlate the behavioral outcome with electrophysiological responses. Figure 4A illustrates the experimental setup used to perform the in vivo unitary extracellular recordings of spinal WDR cells. In this figure, the recordings correspond to the baseline neuronal response (t = 0) elicited by twenty electrical pulses given in the paw receptive field. All neurons recorded in the present experiments were found at an average of 700 ± 192 μm from the spinal surface. As illustrated in Figures 4B,C, the peripheral electrical stimulation produces a well-defined and stereotyped WDR cell response. In general, under our parameters of electrical stimulation used (0.2 Hz; 1 ms pulse duration), we observed that although some cells exhibited high post-discharge events (see Cell 1), an inconsistent wind-up was elicited (calculated accordingly to the formula given by Gjerstad et al., 1996). As previously reported, wind-up is considered as a facilitation of neural discharge evoked by repetitive stimulation of primary afferent fibers (Mendell, 1966). This process elicited in WDR cells seems to mainly depend on the frequency stimulation, and in    Frontiers in Pharmacology frontiersin.org general, frequencies >0.5 Hz are required to consistently produce a wind-up (Mendell, 1966;Dickenson and Sullivan, 1987;Chapman and Dickenson, 1992;Li et al., 1999;You et al., 2003); in this sense, 0.2 Hz seems not to be enough to recruit a facilitatory input leading to wind-up. Furthermore, at the basal time (t = 0), the mean average evoked potential of all animals tested was 667 ± 29 spikes. Since the evoked potential did not follow a normal distribution along the experimental groups, a non-parametric test was used, and we found no statistically significant difference (see Figure 4G; Table 2 for details). The following results were normalized to perform a parametric statistical analysis.

General effects of peripheral electrical stimuli on wide dynamic range cell responses
Since we recorded spinal WDR cells for 60 min, analyzing the effect of time on the neuronal responses was crucial to exclude a time-dependent effect. The one-way ANOVA suggests that no time-dependent changes in neuronal responses occurred during our experimental protocols. Specifically, in the control group, time had no effect on Aδ-(F  Figure 5 shows the raw tracing of a single WDR neuron cluster response, the raster plots, and the PSTHs before and after different treatments. Figure 5A shows the PSTHs obtained during the control condition, whereas Figures 5B,C exemplify the neural activity of second-order WDR cells before (basal response) and after clonidine treatment. In contrast to the control group ( Figure 5A), spinal clonidine, particularly at 10 nmol ( Figure 5C), diminished the neuronal firing responses (events) elicited by the RF electrical stimulation. We must clarify that each PSTH represents the neuronal evoked activity of one WDR cell induced by twenty electrical pulses; these evoked neuronal events could be broken down according to the conduction velocities of the primary afferent fibers (i.e., Aβ-, Aδ-, and C-fibers).

Spinal clonidine-induced electrophysiological antinociception is reversed by BRL 44408
In this set of experiments, we found that BRL 44408 reversed the antinociception induced by clonidine. Figure 6A shows an example of a raw tracing of a single WDR neuron cluster response, the raster plots, and PSTH obtained in an animal pre-treated with BRL 44408 before clonidine administration. Note that BRL 44408 blocked the clonidine-induced diminution of WDR neuronal activity.
Upon quantifying the data from the PSTHs, we found that spinal clonidine decreases the firing response elicited by electrical stimuli on the RF (Figures 6B-I). Specifically, clonidine has no substantial effect on the neuronal activity associated with Aβ-fiber activation ( Figure 6B), but we observed a potent inhibition of neuronal activity related to the activation of Aδ-( Figure 6C) rather than C-fibers ( Figure 6D). This antinociceptive effect started 10 min after clonidine administration, peaked at 40-50 min and lasted up to 60 min as previously reported Bonnet et al., 1989;Murata et al., 1989). Furthermore, as expected, the clonidine-induced inhibition of nociceptive Aδ-and C-fiber activity was reversed by spinal pre-treatment with BRL 44408 (1 nmol). In addition, as shown in Figure 6E, the postdischarge activity was reduced by 10 nmol clonidine, an Frontiers in Pharmacology frontiersin.org effect reversed by BRL44408. Analyzing these results as global neuronal activity ( Figures 6F-I), we corroborated that clonidine selectively blocks the neuronal activity associated with the activation of primary nociceptive fibers, and this effect is abolished by a selective α 2A -adrenoceptor antagonist (BRL 44408). Together, these results highlight the relevance of primary afferent fibers innervating second-order WDR cells in clonidine-induced antinociception.

Spinal JP 1302 does not influence the neuronal activity of second-order wide dynamic range cells
Based on the behavioral results, where 1 nmol JP 1302 per se inhibited the flinches evoked by formalin ( Figures 3D-F), we tested the effect of JP 1302 on WDR neuronal activity ( Figures  7A-F). The results showed that spinal JP 1302 (1 or 10 nmol) did not affect the peripheral evoked neuronal activity of Aβ-, Aδ-, C-fibers and post-discharge ( Figures 7C-F). These results may imply that the mechanisms involved in the antinociception induced by JP 1302 are not mediated by an action on WDR neurons or primary afferent fibers innervating these secondorder cells.

JP 1302-induced behavioral antinociception is blocked by spinal bicuculline
On this basis and considering that some evidence suggests that α 2C -adrenoceptors decrease GABAergic transmission (see discussion section), we hypothesized that the JP 1302-induced behavioral antinociception could be indirectly mediated by a spinal GABAergic mechanism. Accordingly, i.t., administration of bicuculline (0.3 nmol; a In this case, and as previously reported in the formalin test (Dirig and Yaksh, 1995;Peng et al., 2015;Ryu et al., 2021) bicuculline per se does not influence flinching behavior.

General
This study was designed to pharmacologically dissect the role of different α 2A/2B/2C -adrenoceptor subtypes in clonidine-induced antinociception. Using tonic (i.e., formalin test) or acute (i.e., peripheral electrical stimulation) nociceptive stimuli, we show that clonidine-induced antinociception is mediated by α 2A -but not α 2B/2C -adrenoceptor activation. Given that in the electrophysiological experiments, clonidine inhibited the neuronal activity associated with electrical activation of Aδ-and C-fibers (but not Aβ-fibers), a presynaptic effect on nociceptive primary afferent fibers (PAFs) is supported. Furthermore, the ongoing firing (post-discharge) was attenuated by clonidine; an effect also reversed by the BRL 44408. Besides, we found that spinal JP 1302 (a selective α 2C -adrenoceptor antagonist) induced per se behavioral but not electrophysiological antinociception, an effect reversed by bicuculline (a preferential GABA A receptor blocker). Apart from the implications discussed below, our data support the notion that: 1) antinociception (acute or tonic) induced by clonidine is mediated by α 2A -adrenoceptors, whereas 2) pharmacological blockade of α 2C -adrenoceptors during tonic nociception elicits antinociception, thus, unmasking a potential pronociceptive action of this receptor (Figure 8).
One important thing in the experimental protocols followed was that the effect of clonidine was analyzed during 1 h. This experimental design was based on the long-lasting antinociception induced by clonidine when given by a spinal route, despite the half-life in the spinal cord being~30 min (Castro and Eisenach, 1989;Khan et al., 1999). Accordingly, seminal reports have consistently shown that this compound induces a spinal long-lasting (>1 h) analgesia/ antinociception peaking after 30 min post-injection Bonnet et al., 1989;Murata et al., 1989;Khan et al., 1999). At first glance, we could hypothesize that this long-lasting effect could be partly mediated by the engagement of supraspinal mechanisms enhancing a descending inhibitory pathway. Indeed, we must acknowledge that activation of the endogenous noradrenergic descending pathway plays a key role in spinal pain modulation in naïve and neuropathic rodents (Patel et al., 2018). Nevertheless, Murata et al. (1989) showed in WDR cell recordings that antinociception elicited by spinal clonidine was shows the raw tracing of a single second-order WDR neuron cluster response induced by one electrical stimulus-evoked in Cell 1 (left) or Cell 2 (right). (E) shows the raster plot and (F) peri-stimulus time histograms (PSTH) constructed from twenty WDR neuronal responses to RF stimulation depicting the different fiber components (Aβ-, Aδ-, C-fibers, and post-discharge). Note that, under our experimental condition, no clear wind-up is elicited. (G) shows that the basal response, i.e., the total of action potentials evoked at t = 0 in the different experimental groups, is not statistically different (χ 2 = 4.535; p = 0.605).
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FIGURE 5
Spinal clonidine selectively blocks the nociceptive neuronal firing of WDR cells. (A-C) depict the raw tracing of a single second-order WDR neuron cluster response induced by one electrical stimulus (upper tracing), the raster plot (middle), and peri-stimulus time histograms (PSTH) constructed from 20 WDR responses to receptive field (RF) electrical stimulation before (basal) and after treatment. In accordance with the fiber conduction velocities, the panels in the figure depict the different fiber components (Aβ-, Aδ-, C-fibers, and post-discharge) of the spinal WDR cell response. Note that clonidine (1 and 10 nmol) diminished the neuronal activity; this clonidine-induced inhibition (observed as a diminution of events) is mainly associated with neural action on nociceptive fibers (i.e., Aδ-and C-fibers) and ongoing (post-discharge) activity, whereas the firing of Aβ-fibers remains unaltered.
Frontiers in Pharmacology frontiersin.org 12 unaffected in animals spinally transected. Consequently, it seems that the effect of this ligand (when given spinally) mainly relies upon spinal cord level.
In this regard, the effect of spinal clonidine on WDR cells was tested to give an electrophysiological correlate. At the spinal level, it is well-known (Urch and Dickenson, 2003) that these secondorder neurons receive concomitant input from PAFs, and

FIGURE 6
The role of spinal α 2A -adrenoceptors in clonidine-induced electrophysiological antinociception. In (A), a pair of raw tracings of a single secondorder WDR neuron cluster response induced by one electrical stimulus (upper tracing), the raster plot (middle), and peri-stimulus time histogram (PSTH) depicting the effect of BRL 44408 on the clonidine-induced antinociception. Notice that under α 2A -adrenoceptors blockade with BRL 44408 (1 nmol), the antinociceptive action of clonidine is not elicited. (B-E) show the time-course changes in the percentage average of the different fibers activating WDR cell responses and post-discharge elicited by receptive field (RF) electrical stimulation and the effects of spinal clonidine (1 and 10 nmol/10 μl; n = 5 each dose). Notice that 1 nmol clonidine only inhibits the neuronal firing of Aδ-fibers (C), while 10 nmol clonidine inhibits the activity of Aδ-fibers (C), C-fibers (D), and post-discharge (E). (F-I) show the global neuronal activity (obtained from the respective time course figures) of Aβ-, Aδ-, C-fibers, and post-discharge in response to spinal clonidine. Clonidine preferentially blocks the neuronal activity associated with Aδ-, C-fibers, and post-discharge but not Aβ-fibers. On the other hand, blockade of the α 2A -adrenoceptors with BRL 44408 (1 nmol) inhibits the clonidine-induced electrophysiological antinociception.
Frontiers in Pharmacology frontiersin.org electrical stimulation of the peripheral RF produces a triphasic response corresponding to the activation of Aβ-, Aδ-and C-fibers (Figure 4), also a post-discharge can be elicited (Figure 4). This experimental approach let us analyze the impact of clonidine in nociceptive transmission, particularly how this ligand affects the firing of PAFs. As illustrated in the PSTHs of WDR cells ( Figure 5) or the temporal course of neuronal activity (Figure 6), 10 nmol clonidine preferentially diminishes the neuronal firing associated with activation of nociceptive Aδ-and C-fibers, supporting the notion that spinal clonidine-induced inhibition via presynaptic action (i.e., on PAFs). In addition, the ongoing activity (i.e., the postdischarge) was also inhibited by 10 nmol clonidine. In direct support of these findings, in vivo (Sonohata et al., 2004) and in vitro (Pan et al., 2002;Kawasaki et al., 2003) electrophysiological recordings show that α 2 -adrenoceptor activation diminished the excitatory postsynaptic current evoked by PAF stimulation. At this point, we need to remember that post-discharge represents a late response of C-fibers to peripheral stimulation that can be endured by a

FIGURE 7
Spinal JP-1302 does not affect wide dynamic range (WDR) cell activity, but behaviorally, it inhibits nociception by increasing GABAergic activity.
(A,B) depict the peri-stimulus time histogram (PSTH) constructed from 20 WDR responses to receptive field (RF) electrical stimulation before (basal) and after treatment (vehicle or JP 1302); also, in these panels, the raster plot and raw tracing of a single second-order WDR neuron cluster response elicited by one electrical stimulus (upper tracing) are illustrated. In accordance with the fiber conduction velocities, the panels in the figure depict the different fiber components (Aβ-, Aδ-, and C-fibers) of the spinal WDR cell response. Note that JP 1302 seems not to exert an effect on the WDR neuronal activity; indeed, when the data are analyzed as a percent of change of the neuronal activity (C-F), JP 1302 does not impact (statistically) the neuronal firing associated with the activation of Aβ-, Aδ-, C-fibers, and post-discharge. (G) shows the effect of intrathecal (i.t.) injection of 0.3 nmol bicuculline (a preferent GABA A receptor blocker) on the JP 1302-induced inhibition of the flinches induced by formalin (1%; 50 μl, s.c.) during phase I (P1) and phase II (P2). Note that bicuculline per se did not affect formalin-induced flinching behavior, but this GABA A channel blocker reversed the effect of JP 1302. (H,I) represent the data as the area under the mean number of flinches against the curve (AUC) and show that bicuculline inhibited the JP 1302-induced antinociception in both phases (P1, P2).
Frontiers in Pharmacology frontiersin.org traumatic injury leading to a neuronal hyperexcitability observed electrophysiologically as a wind-up (Woolf, 1996). However, under our experimental approach, we were unable to induce wind-up. Hence, an interesting question not addressed in the present work relates to how wind-up can be affected by noradrenergic transmission. Accordingly, to the behavioral experiments, we also showed that presynaptic pharmacological blockade of α 2A -adrenoceptors in WDR recordings is relevant to the effect of clonidine. Although these data agree with histological/molecular data showing the presence of α 2A -adrenoceptors at the PAF level (i.e., Aδ-and C-fibers) (Stone et al., 1998;Birder and Perl, 1999), no direct in vivo evidence about a functional role of the presynaptic α 2Aadrenoceptor subtype had been previously reported.
Regarding α 2B -adrenoceptors, the behavioral experiments reject the involvement of this adrenoceptor subtype. As shown in Figures 3A-C supramaximal dose of imiloxan (α 2Badrenoceptor antagonist) does not affect clonidine's antinociception. Current literature seems to support the notion that spinal α 2B -adrenoceptor does not influence pain modulation (for refs., see Pertovaara, 2013;Llorca-Torralba et al., 2016;Bahari and Meftahi, 2019). For example, in an inflammatory pain model, Zhang et al. (2012) showed that spinal α 2B -adrenoceptor blockade with imiloxan does not reverse the antinociception induced by enhancement of the descending noradrenergic tone, suggesting that this receptor subtype is not necessary for the noradrenergic analgesic effect. However, in neuropathic pain models involving ligature of spinal

FIGURE 8
Proposed mechanisms implying a differential role of the α 2A -and α 2C -adrenoceptors subtypes in pain modulation at the spinal cord level. In general, α 2 -adrenoceptors are coupled to G i/o proteins; thus, when activated, they could promote the inhibition of neurotransmitter release by inhibiting adenylate cyclase activity and inactivating Ca 2+ channels and/or facilitation of K + currents. Our behavioral and electrophysiological data support the contention that clonidine (a non-selective α 2A/2B/2C-adrenoceptor agonist) induces antinociception via activation of α 2Aadrenoceptors, probably located at presynaptic level (in the primary nociceptive Aδ-and C-fibers), causing a diminution of the WDR cell nociceptive firing. Indeed, α 2A-adrenoceptors activation inhibits tonic (formalin) and acute (electrical) nociceptive input. On the other hand, the selective α 2Cadrenoceptor antagonist, JP 1302, produces per se only behavioral but not electrophysiological antinociception (measured on second WDR cells), suggesting an inhibitory effect on tonic nociception. Since JP 1302-induced antinociception was blocked by bicuculline, a GABAergic mechanism has been resembled. The data could be interpreted as follows: (i) presynaptic activation of α 2A -adrenoceptors by clonidine inhibits acute and tonic peripheral nociception; (ii) the JP 1302-induced antinociception is not mediated by inhibition of the nociceptive primary afferent fibers activity; and (ii) considering that α 2C -adrenoceptors can be found in GABAergic neurons, we could suggest that under a tonic nociceptive stimulus (e.g., inflammatory), activation of α 2C -adrenoceptors play a pronociceptive role. Hence (see Discussion for details), the data support the notion that α 2Aadrenoceptor are mainly localized in primary nociceptive afferent fibers modulating the activity of WDR cells. In contrast, α 2C -adrenoceptors seem to be present in spinal interneurons rather than capsaicin-sensitive fibers modulating second-order nociceptive specific cells.
Frontiers in Pharmacology frontiersin.org nerves (not tested in the present experiments) and using selective ligands, it seems that α 2B -adrenoceptor acquire a role in the inhibition of nociception (Chu et al., 2015;Rodríguez-Palma et al., 2022) an effect probably related with cortical activation of this receptor subtype rather than spinal mechanism (Chu et al., 2015). In essence, our data suggest that spinal α 2B -adrenoceptor does not play a role in tonic inflammatory and acute nociception. In contrast, current data about the functional role of α 2Cadrenoceptors inhibiting nociception is scarce, and as discussed by Pertovaara (2013), the impact of this receptor subtype in spinal nociception is unclear. These inconsistent data are partly due to the lack of selective compounds used to dissect the contribution of different α 2A/2B/2C -adrenoceptor subtypes. For example, in transgenic mice, Fairbanks et al. (2002) showed that α 2C -adrenoceptors have a subtle role in the moxonidine (a mixed I 1 imidazoline/α 2C -adrenoceptor agonist)-induced antinociception, whereas Malmberg et al. (2001) suggest that α 2A -but not α 2C -adrenoceptors are indispensable for the antinociceptive action of i.t., dexmedetomidine (a nonselective α 2A/2B/2C -adrenoceptor antagonist).
In our behavioral experiments, we used JP 1302, a selective α 2C -adrenoceptor antagonist (Sallinen et al., 2007;Proudman et al., 2022), to test the role of this receptor in clonidine-induced antinociception. According to Figures 3A-C i.t., JP 1302 was unable to block clonidine's effect. Similar results were found in a neuropathic pain model (Rodríguez-Palma et al., 2022) or writhes-induced by sleep deprivation (Yaoita et al., 2018), showing that systemic JP 1302 does not preclude the behavioral antinociceptive action of α 2 -adrenoceptor agonists (ST-91 or tizanidine given systemically). However, when we analyzed the per se effects of the antagonist ( Figures 3D-F), we found that in sharp contrast to BRL 44408 or imiloxan, i.t., JP 1302 diminishes the number of flinches induced by formalin (in phase I and II), implying that this antagonist has an antinociceptive effect during the formalin test.

The potential pronociceptive role of α 2C -adrenoceptors in tonic nociception
In contradiction to the behavioral data, when we explored the effect of JP 1302 using an electrophysiological approach (Figures 7A-F), no effect was observed in the neuronal firing of WDR cells. This discrepancy could be attributable to the nature of the noxious stimulus used (i.e., tonic vs. acute) and may imply that JP 1302 exerts its antinociceptive action in a different way than clonidine not involving presynaptic or WDR cell inhibition. Admittedly, an interesting iteration to try to disentangle and give an electrophysiological correlate would have been to analyze the effect of these ligands on wind-up, taking into account that this neuronal process has been related with central sensitization due to recruitment of nociceptive circuits beyond of Aδ-and C-fibers activation. Regardless, if we consider that this compound exerts an antinociceptive effect via the blockade of α 2C -adrenoceptors, it is interesting to note that α 2C -adrenoceptors have been localized in non-noradrenergic brainstem descending fibers and postsynaptic sites in spinal interneurons (Stone et al., 1998;Olave and Maxwell, 2002). To our knowledge, no report about an antinociceptive per se action of selective α 2C -adrenoceptor antagonists exist. Consequently, the question is: How can α 2C -adrenoceptor blockade induce spinal antinociception? Proudman et al. (2022) suggest that the selectivity of JP 1302 is reliable for dissecting between the α 2A/2B/2C -adrenoceptor subtypes (see Table 1), but this antagonist also displays some affinity for α 1A -adrenoceptors (pK i :6.2). Hence, we cannot ignore that interaction between JP 1302 and α 1A -adrenoceptors could exist. Certainly, using an optogenetic approach, Kucharczyk et al. (2022) showed that activating a discrete noradrenergic descending pathway from locus coeruleus provokes a diminution of WDR activity, an effect blocked by prazosin (a non-selective α 1A/1B/1D -adrenoceptor antagonist), suggesting a role for α 1 -adrenoceptors. However, in the formalin test, i.t., prazosin does not affect nocifensive behavior (Jeong et al., 2011;Park et al., 2011). Similarly, the clonidine-induced inhibition of WDR responses evoked by NMDA was unaffected by prazocin (Zhang et al., 1998). Indeed, subcutaneous formalin injection tends to decrease the [ 3 H]-prazosin binding sites in the spinal cord (Nalepa et al., 2005), suggesting that under an inflammatory stimulus, the probability that this receptor could be exerting a pharmacological effect is minimal. Besides, using an acute (thermal) or neuropathic pain model (L 5 -L 6 nerve ligation), i.t., α 1 -adrenoceptor agonist (methoxamine) does not have any impact on the pain threshold (Nagasaka and Yaksh, 1990;Yaksh et al., 1995). Together these data support our contention that under our experimental conditions, the effect of JP-1302 may be mediated by its interaction with the α 2C -adrenoceptor.
If spinal α 2C -adrenoceptor blockade produces an antinociceptive effect in the formalin test but not in the electrophysiological experiments, the most straightforward interpretation of these findings may suggest (but does not prove) that under tonic nociception α 2C -adrenoceptor activation counter-balance the antinociceptive effect of descending noradrenergic system. In this regard, subcutaneous formalin induces not only an increase in the endogenous descending noradrenergic activity (Ma et al., 2001;Sajadeianfard et al., 2005;Martins et al., 2013) but also a rise in the mRNA expression of α 2C -adrenoceptor at spinal cord level . Coupled with the evidence suggesting that this receptor is expressed in GABAergic interneurons (Holmberg et al., 1999;Olave and Maxwell, 2002), we hypothesize that during spinal tonic nociceptive input, activation of this receptor could, in turn, diminish the GABAergic transmission, favoring a pronociceptive state. This hypothesis gain weight Frontiers in Pharmacology frontiersin.org considering that at the striatum level, it has been suggested that activation of α 2C -, but not α 2A -adrenoceptors inhibits GABA release (Holmberg et al., 1999;Zhang and Ordway, 2003). Hence, i.t., JP 1302 indirectly favors an antinociceptive effect by improving the activity of GABAergic neurons. To prove this hypothesis, and using the formalin test, we assessed the effect of spinal bicuculline (unspecific antagonist of the GABA A receptors) on the JP 1302-induced antinociception. At this point, we must emphasize that 0.3 nmol (0.11 μg) bicuculline per se does not influence flinching behavior induced by formalin (Figures 6F-H). This data agrees with previous reports showing that i.t., <0.3 μg bicuculline (<0.8 nmol) does not impact this nocifensive behavior (Dirig and Yaksh, 1995;Peng et al., 2015;Ryu et al., 2021). However, although some reports showed that spinal bicuculline is pronociceptive, this effect depends on the dose of bicuculline and the explored pain model. In the case of the formalin test, Kaneko and Hammond (1997) showed that at formalin concentrations <1%, an increase of flinches is induced by bicuculline, particularly at 0.3 μg (i.e., 0.8 nmol). Therefore, under our experimental conditions (1% formalin), the concentration used of bicuculline (0.3 nmol equivalents to 0.11 μg) seems to be adequate to evaluate the role of GABAergic participation in the JP 1302 effect.
The data show that the antinociception induced by JP 1302 was reversed with 0.3 nmol bicuculline ( Figures 7H-J), suggesting that JP 1302 favors GABAergic transmission. In vitro evidence indicates that GABAergic neurons in the substantia gelatinosa can be hyperpolarized by noradrenaline (Hantman and Perl, 2005). Specifically, by recording GABAergic neurons at the superficial dorsal horn level using the patch-clamp technique, Gassner et al. (2009) proved that although the main effect of noradrenaline on GABAergic neurons is depolarization, a minor proportion of these cells are hyperpolarized via α 2adrenoceptors. In these studies, the specific receptor subtype was not identified. Together, these data support our contention that by inhibiting GABAergic neurotransmission, α 2C -adrenoceptors may play a pronociceptive role (particularly during tonic nociception). The pronociceptive role of this receptor has also been suggested in in vitro assays by Chen et al. (2008), where they showed that α 2C -adrenoceptor activation inhibits opioid release in the spinal cord; thus, blocking this receptor would induce antinociception.

A final consideration about the role of spinal α 2 -adrenoceptors subtypes in pain modulation
Our data suggest that during tonic pain, both α 2A -and α 2C -adrenoreceptors are activated, and one question that could arise is: exist a particular situation where α 2C -surpass the antinociceptive effect of α 2A -adrenoreceptors? Current evidence shows that the net impact of non-selective α 2 -adrenoceptor agonists is antinociception in acute and neuropathic pain conditions (for refs. see Bahari and Meftahi, 2019), pointing out the relevance of α 2A -over α 2C -adrenoreceptors. Certainly, we need to keep in mind that plastic changes in the spinal α 2 -adrenoreceptors occur under neuropathic pain and may explain, at least in part, the variability in the efficacy of α 2 -adrenoreceptor agonists depending on the stage of neuropathic pain . At this point, it is worth mentioning that the expression of the different α 2A/2B/2C -adrenoceptor subtypes at the spinal cord level under pathological pain has been analyzed. Stone et al. (1999) showed that after spinal nerve ligation, the immunostaining of α 2Aadrenoceptor is reduced, whereas α 2C -adrenoceptor is enhanced. Hayashida and Eisenach (2010) discussed the physiological relevance of this change, suggesting that under peripheral nerve injury, the function of α 2adrenoceptors can be altered. Furthermore, the relevance of α 2C -adrenoreceptor in spinal nociception will be answered with a selective α 2C -adrenoreceptor agonist (not available yet) or the use of more precise approaches (e.g., opto-and chemogenetics). Regardless, at the spinal level, the effect of clonidine in healthy and pathological pain is analgesia. Therefore, our results deserve further investigation.
Finally, at the spinal dorsal horn level, the role of noradrenergic transmission is far more complex than initially conceived. This complexity may depend on the type of adrenergic receptor stimulated, the localization of the receptor (e.g., PAFs, interneurons, second-order neurons), and the type of nociceptive input. For example, at the spinal trigeminal level, both α 2A -, and α 2Cadrenoceptors seem to inhibit nociceptive transmission (Villalón et al., 2012). More recently, it has been suggested that at the spinal cord level, the noradrenergic system elicits a fine-tunning of nociception by activation of α 1 -or α 2adrenoceptors; indeed, they proposed that spinal α 2adrenoceptors located presynaptically in the noradrenergic projections from the ventral LC can be modulating the firing of GABAergic interneurons (see Suppl Fig 2 in Kucharczyk et al. (2022)). Furthermore, as illustrated by Hickey et al. (2014), optogenetic experiments demonstrate that stimulation of different regions of locus coeruleus can evoke an antinociceptive or pronociceptive spinal effect; in this case, although i.t., atipamezole (a non-selective α 2A/2B/2Cadrenoceptor antagonist) blocked the spinal clonidineinduced antinociception, this antagonist also seems to enhance the antinociceptive action of optogenetic stimulation of the noradrenergic system in the LC, but the mechanism/receptor by which atipamezole enhances antinociception was not further explored. This divergent effect of noradrenergic transmission on pain modulation has also been observed in healthy humans, where activation Frontiers in Pharmacology frontiersin.org of α 2 -adrenoceptors (by yohimbine) elicits both antinociceptive and pronociceptive actions (Vo and Drummond, 2015).

Conclusion
Taken together, our data suggest that spinal α 2A -and α 2Cadrenoceptors exert a differential (opposite) effect on nociceptive transmission. Specifically, α 2A -adrenoceptor activation on PAF inhibits the tonic and acute nociceptive input, whereas α 2Cadrenoceptor activation appeared to inhibit GABAergic transmission, favoring nociception during a tonic (inflammatory) stimulus.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

Ethics statement
The animal study was reviewed and the protocol was approved by the Institutional Animal Care and Use Committee at the Instituto de Neurobiología, UNAM.


Single-cell analysis of Kaposi’s sarcoma-associated herpesvirus infection in three-dimensional air-liquid interface culture model

The oral cavity is the major site for transmission of Kaposi’s sarcoma-associated herpesvirus (KSHV), but how KSHV establishes infection and replication in the oral epithelia remains unclear. Here, we report a KSHV spontaneous lytic replication model using fully differentiated, three-dimensional (3D) oral epithelial organoids at an air-liquid interface (ALI). This model revealed that KSHV infected the oral epithelia when the basal epithelial cells were exposed by damage. Unlike two-dimensional (2D) cell culture, 3D oral epithelial organoid ALI culture allowed high levels of spontaneous KSHV lytic replication, where lytically replicating cells were enriched at the superficial layer of epithelial organoid. Single cell RNA sequencing (scRNAseq) showed that KSHV infection induced drastic changes of host gene expression in infected as well as uninfected cells at the different epithelial layers, resulting in altered keratinocyte differentiation and cell death. Moreover, we identified a unique population of infected cells containing lytic gene expression at the KSHV K2-K5 gene locus and distinct host gene expression compared to latent or lytic infected cells. This study demonstrates an in vitro 3D epithelial organoid ALI culture model that recapitulates KSHV infection in the oral cavity, where KSHV undergoes the epithelial differentiation-dependent spontaneous lytic replication with a unique cell population carrying distinct viral gene expression.

The oral cavity is the major site for transmission of Kaposi's sarcoma-associated herpesvirus (KSHV), but how KSHV establishes infection and replication in the oral epithelia remains unclear. Here, we report a KSHV spontaneous lytic replication model using fully differentiated, three-dimensional (3D) oral epithelial organoids at an air-liquid interface (ALI). This model revealed that KSHV infected the oral epithelia when the basal epithelial cells were exposed by damage. Unlike two-dimensional (2D) cell culture, 3D oral epithelial organoid ALI culture allowed high levels of spontaneous KSHV lytic replication, where lytically replicating cells were enriched at the superficial layer of epithelial organoid. Single cell RNA sequencing (scRNAseq) showed that KSHV infection induced drastic changes of host gene expression in infected as well as uninfected cells at the different epithelial layers, resulting in altered keratinocyte differentiation and cell death. Moreover, we identified a unique population of infected cells containing lytic gene expression at the KSHV K2-K5 gene locus and distinct host gene expression compared to latent or lytic infected cells. This study demonstrates an in vitro 3D epithelial organoid ALI culture model that recapitulates KSHV infection in the oral cavity, where KSHV undergoes the epithelial differentiation-dependent spontaneous lytic replication with a unique cell population carrying distinct viral gene expression.

Author summary
While KSHV is the etiologic agent of Kaposi's sarcoma that frequently occurs in AIDS patients, there is not biologically relevant, reproducible in vitro culture system to study KSHV infection and spontaneous lytic replication. Here, we describe that a 3D human oral epithelial ALI culture that can support a robust KSHV infection and spontaneous lytic replication associated with oral epithelial differentiation. scRNAseq analysis identified considerable alteration of host gene expression in both KSHV-infected cells and Introduction KSHV is a gamma herpesvirus and the etiologic agent of Kaposi's sarcoma (KS), primary effusion lymphoma, and multicentric Castleman's disease [1][2][3]. Like other herpesviruses, KSHV switches between the latent and lytic lifecycles [4]. Latent KSHV is characterized by limited viral gene expression, dependency on the host for replication of the viral episome, and co-segregation of the viral episome with host chromosomes during mitosis. KSHV defaults to this quiescent, latent phase to evade the host immune system and maintain a persistent, lifelong infection. In response to an external stimulus such as oxidative stress, hypoxia, and certain chemicals, the latent KSHV lifecycle can be disrupted and enter lytic reactivation [5].
The oral cavity provides a favorable environment for the virus to reactivate upon external stimuli. Previous studies have shown that several infectious pathogens, including Staphylococcus aureus, stimulate lytic reactivation in KSHV-infected oral cells through their pathogenassociated molecular patterns (PAMPs) and host Toll-like receptor (TLR) signaling [6]. Moreover, intensive epigenetic reorganization during oral epithelial differentiation may trigger KSHV reactivation [7]. In addition, the oral cavity sheds infectious KSHV virions into the saliva two to three log folds higher in titer compared to other anatomic sites [8], which is why oral transmission is considered as the major route of KSHV transmission [9]. However, the molecular mechanism behind how KSHV establishes infection in the oral cavity and how lytic reactivation is triggered in the oral epithelial cells remains unclear.
The oral buccal mucosal epithelium serves as a barrier between the underlying oral tissues and the outside environments such as the soft palate, cheeks, and floor of the mouth [10]. The composition of human oral epithelial barrier is complex, with a structure consisting of three major layers: basal, intermediate, and superficial. The basal layer consists of mitotically active epithelial cells connected to neighboring cells through desmosomes at the bottom of the epithelium [11]. These cells are characterized by the expression of Keratin 5 (KRT5), Keratin 14 (KRT14), and S100A2 [12][13][14]. As the basal cells differentiate upwards into the intermediate layer, they downregulate the expression of KRT5, KRT14, and S100A2, and express a new set of genes such as Keratin 13 (KRT13), NOTCH3, S100A4, and TGM5 [11,12,[14][15][16]. As these cells differentiate into the superficial or cornified layer, they undergo another major transcriptional change in order to deposit glutamine-and lysine-rich proteins such as SPRR2A and SPRR2E [15] on the inner surface of the plasma membrane to be crosslinked with the keratin cytoskeleton with an influx of Ca 2+ that also upregulates S100 proteins such as S100P [14]. These three layers ultimately form oral epithelial tissues for the protection of the organism from chemical, microbial, and physical challenges.
While 2D monolayer cultures of oral epithelial cell-based infections have been extensively studied, they are limited by the lack of physiological relevance in mimicking the epithelial barrier. A deeper understanding of KSHV de novo infection and its spontaneous reactivation require comprehensive characterization of virus infection in multiple epithelial cell subtypes. In vitro epithelial air-liquid interface (ALI) culture models are highly relevant tools that are able to complement animal models for understanding the pathological mechanism of KSHV infection [17]. While these ALI models mimic human oral epithelial tissue and demonstrate KSHV reactivation during epithelial cell differentiation [17,18], de novo infection of KSHV in this model has not been reported.
Using 3D oral epithelial ALI cultures fully differentiated from healthy oral keratinocytes, we developed a novel de novo KSHV infection model. We found that exposing the basal layer was necessary for initial KSHV infection and confirmed that epithelial differentiation tightly correlated with KSHV lytic reactivation. To further understand the effect of KSHV on host gene expression in oral epithelial tissues, we leveraged scRNAseq to profile the gene expression across all cells in the different epithelial layers. We identified that host gene expression in infected cells, as well as uninfected cells, were drastically changed by presence of KSHV, thereby altering keratinocyte differentiation and cell death. Moreover, we discovered a distinct population of infected cells with a limited early lytic KSHV gene expression and unique host gene expression profile compared to latent or lytic cells. Taken together, our proposed de novo KSHV infection model demonstrates a physiologically relevant model that closely mimics KSHV infection in the oral cavity and provides insight into the early stages of KSHV infection in oral transmission.

KSHV infection of primary oral epithelial ALI culture undergoes spontaneous replication
3D oral epithelial tissues in ALI culture are 8-11 cells thick with differentiated basal, intermediate, and superficial epithelial layers. Intact 3D oral epithelial tissues were initially infected from the apical side with rKSHV.219 that contains green fluorescent protein (GFP) gene indicating KSHV infection and red fluorescent protein (RFP) gene under the control of the PAN RNA promoter indicating KSHV lytic replication [19] (Fig 1A). Since KSHV failed to establish a productive infection in an intact 3D oral epithelial tissue, we introduced scoring to disrupt the epithelial barrier with (deep) or without (light) basal exposure for KSHV infection (S1A Fig). When these wounded tissues were incubated with either Dulbecco's phosphate-buffered saline (DPBS, Mock) or infectious rKSHV.219, KSHV was able to establish an infection upon deep wounding-induced exposure of the basal epithelial cells (S1B Fig). Immunohistochemistry and immunofluorescence assays also revealed that cells around wounded area expressed LANA and ORF65 (Fig 1B and 1C) and that GFP+ and RFP+ cells were co-stained with LANA and ORF59, indicating GFP and RFP signals as surrogate markers for KSHV infection (S1C Fig). Interestingly, KSHV entry receptors integrin β1 and CD98 were primarily expressed in the basal epithelial cells (S1D Fig), suggesting that integrin β1 and CD98 expression in the basal layer allows efficient KSHV infection [20,21].
While primary normal human oral gingival keratinocytes (HOKg) were highly susceptible to KSHV infection (GFP+) in 2D culture, only a minor population [~3% at 6 days post infection (dpi)] of KSHV-infected cells underwent spontaneous lytic replication (RFP+) (Figs 1D, 1F and S2). In contrast, a large portion (~45% at 6 dpi) of KSHV infected basal epithelial cells in 3D organoids underwent spontaneous lytic replication (RFP+) (Figs 1E, 1F and S2). Moreover, KSHV-infected 3D epithelial cultures showed significantly increased expression of RTA (immediate early), K2 (early), and ORF25 (late) across all lytic stages ( Fig 1G). Finally, while viral copy number decreased over time in the supernatants of 2D culture (Fig 1H, left), it rapidly increased in the supernatants of 3D tissue culture, suggesting virion release (Fig 1H,  right). This study of primary oral 3D epithelial ALI cultures demonstrates that KSHV readily infects the basal epithelial layers induced by deep wounding and undergoes a rapid transition from latency to spontaneous lytic replication to potentially release virions, which may recapitulate in vivo human oral KSHV infection.

PLOS PATHOGENS
Single-cell analysis of KSHV infection in 3D ALI culture model

Single-cell RNA sequencing reveals enriched lytic KSHV-infected cells in the superficial epithelia
To gain a better understanding of KSHV-infected cells undergoing spontaneous reactivation, we performed scRNAseq on infected 3D epithelial tissues. 3D epithelial tissues established apparent infection around 3 dpi and increased spontaneous reactivation throughout the culture (S3A Fig). Since 3D oral epithelial tissues lost structural integrity at 9 dpi in either mock infection or KSHV infection condition, we selected 3 and 6 dpi of 3D epithelial tissues for scRNAseq analysis. After quality control to remove dying cells (S4A- S4C Fig), we analyzed 36,144 cells harvested from KSHV-infected 3D epithelial tissues and their respective mock controls at 3 and 6 dpi from three independent sets of infection. We used gene expression markers to define the basal layer (KRT5, KRT14, S100A2), intermediate layer (NOTCH3, S100A4, KRT13, TGM5), and superficial layer (S100P, SPRR2A, SPRR2E). Unsupervised clustering showed that cells clustered based on epithelial layer (basal, intermediate, or superficial) and showed progression of epithelial cell differentiation (Fig 2A).
Since the 10X genomics single cell gene expression platform has a limited RNA capture rate of~65%, low abundant viral transcripts such as LANA and RTA were mostly undetected. For this reason, we used GFP and RFP expression as surrogate markers to distinguish the population of virus-infected cells within the infected tissues: for instance, GFP+ and GFP-cells were designated as KSHV-infected cells and bystander cells, respectively ( Fig 1A). Since PAN RNA is the most abundant viral transcript during lytic cycle [22,23], PAN RNA expression was used to further define GFP+/PAN-latent infected and GFP+/PAN+ lytic infected cells (Fig 2A-2C). This showed an enrichment of latent cells in the basal layer (28.57%) at 3dpi and of latent and lytic cells in the superficial layer (36.7% and 39.8%, respectively) at 6dpi (Fig 2B). Lytic cells also expressed higher levels of the superficial layer-specific genes S100P, SPRR2A, and SPRR2E ( Fig 2C). In the cross section of 3D epithelial tissues, GFP+/RFP+ cells were in the superficial layer, while GFP+/RFP-cells were mainly in the basal layer ( Fig 2D). In addition, lytic protein ORF59-and K8.1-expressing lytic cells were mostly present in the superficial exposed areas, while LANA-expressing latent cells were primarily in the basal layer (S3B Fig). This indicated that de novo infection of KSHV occurred only in the basal layer and that KSHV underwent spontaneous reactivation in the superficial layer, where lytic replicating cells were enriched.

KSHV infection dysregulates epithelial differentiation
To characterize epithelial cell differentiation upon KSHV infection, we used immunofluorescence to visualize the epithelial differentiation in mock and KSHV-infected 3D oral epithelial ALI cultures ( Fig 3A). As keratin 5 and keratin 13 are the basal and suprabasal layer markers, respectively [24], keratin 5 was exclusively detected in the basal layer epithelia and keratin 13 was detected throughout the suprabasal layer of mock-infected 3D cultures. In contrast, keratin 5 and keratin 13 were expressed in the basal, suprabasal and superficial epithelial layers in KSHV-infected 3D cultures. Furthermore, since expression of keratins 16 and S100A7 are induced in response to tissue wounding [25,26], their expressions were readily detected in mock-infected 3D culture but weakly detected in KSHV-infected 3D cultures ( Fig 3A). These data suggest that KSHV infection apparently alters epithelial differentiation and wound healing pathways. scRNAseq analysis identified that host gene expression within each epithelial layer was significantly altered upon KSHV infection compared to mock infection ( Fig 3B). Pathway analysis showed that while KSHV infection commonly affected keratinocyte differentiation and protein translation throughout all epithelial layers, it uniquely altered specific pathways at each layer: cell-cell adhesion and response to stress in the basal layer; intermediate filament cytoskeleton organization and acute-phase immune response in the intermediate layer; and cell death and angiogenesis in the superficial layer ( Fig 3C). The intermediate and superficial layers of KSHV-infected tissues showed the upregulated expression of TGM1, IVL, and SFN genes that promote keratinocyte differentiation and the downregulated expression of EPHA2 gene that restricts keratinocyte differentiation ( Fig 3D). Finally, the superficial layer of KSHVinfected tissues showed the upregulated expression of apoptosis inducer RTN4 [27] and the downregulated expression of apoptosis suppressors GADD45B, CFLAR, and BAG1 (Fig 3E). This indicates that KSHV infection dysregulates epithelial differentiation pathway throughout all three layers and host signaling pathways of each layer in a unique way.

Effect of KSHV infection on gene expression of basal epithelia in 3D organoids
When KSHV-infected cells were compared to the uninfected bystander cells in infected 3D epithelial ALI culture, KSHV infection induced more pronounced alterations of cellular gene  Fig 4A). Specifically, KSHV infection in the basal layer led to the upregulation of several host signaling pathways including cell adhesion, cell differentiation, and extracellular matrix organization (Fig 4B). When keratinocyte differentiation-related genes were analyzed within each layer of epithelial organoids upon KSHV infection, they were dramatically dysregulated in KSHV infected cells compared to those in bystander cells (Fig 4C). Specifically, KSHV infected cells highly expressed kallikrein-5 (KLK5) that is involved in the terminal differentiation of keratinocytes, while they showed lower expression of cornulin (CRNN) that is a marker of squamous cells (Fig 4C). Furthermore, immunofluorescence assay also confirmed that compared bystander cells, LANA-positive cells showed higher expression of KLK5, but lower expression of CRNN (Fig 4D and 4E). Loss of CRNN expression has been associated with advanced tumor stage,

PLOS PATHOGENS
Single-cell analysis of KSHV infection in 3D ALI culture model whereas high expression of KLK5 has been positively correlated with tumor invasion [28,29]. This indicates that KSHV infection-mediated alteration of host gene expressions in 3D epithelial ALI culture potentially contributes to viral cancer progression.

A unique cell population with a distinct KSHV gene expression profile
As KSHV exhibits a biphasic lifecycle between latent and lytic infection, we separated KSHVinfected cells into latent (GFP+/PAN RNA-) and lytic cells (GFP+/PAN RNA+ or RFP+) to examine their viral gene expressions (Fig 5A). During de novo infection, approximately 39% and 26% of infected cells showed latency at 3 dpi and 6 dpi, respectively, while 58% of infected cells spontaneously underwent lytic reactivation at 3 dpi and this lytic cell population increased to 66% at 6 dpi (S1 Table). Since the numbers of KSHV-infected cells were low, we combined 3 and 6 dpi timepoints for analyses relating to viral life cycle with the rationale that viral infection stage is the defining factor of infected cells. Interestingly, we found a unique population within the latent group that contained specific lytic gene expression at the KSHV K2-K5 locus and designated these cells as "latent-2" cells (Figs 5A and S3C). Previous work has shown that activating histone modifications are enriched at the K2-K5 locus, accompanied by the transient induction of limited lytic gene expression [30]. The latent-2 population was 1.9% of infected cells at 3 dpi and expanded to 6.8% of infected cells at 6 dpi (Fig 5A, S1 Table). Heatmap analysis of 3 and 6 dpi cells showed that latent-2 cells carried distinct host gene expression pattern from latent and lytic cells (Fig 5B). Pathway analysis showed that a few signaling pathways including viral infection and migration of cells were similarly affected in latent, latent-2, and lytic cells compared to mock-infected cells (Fig 5C). On the other hand, latent-2 cells showed different profiles of several signaling pathways from latent or lytic cells: cell survival, wound healing, HIF1α signaling, ephrin receptor signaling, and NRF2 response

PLOS PATHOGENS
Single-cell analysis of KSHV infection in 3D ALI culture model to oxidative stress were upregulated but apoptosis and PPAR signaling were downregulated (Fig 5D-5E). A recent Landis et al paper [31] also has demonstrated distinct latent lifecycle of KSHV in JSC-1 and BCBL-1 PEL cells that carries a latent-2 like viral gene expression pattern at the KSHV K2 gene region. This reveals the heterogenous nature of KSHV gene expression in oral epithelial 3D organoid upon de novo infection and latent-2 cell population shows a distinct host gene expression profile from latent or lytic cell population.

Discussion
The oral cavity is the major route for KSHV infection and exposure to saliva containing infectious KSHV is primarily responsible for the transmission [8]. Thus, understanding KSHV infection of the oral cavity is essential for the study of viral transmission and pathogenesis. Here, we described a de novo KSHV infection model of fully differentiated 3D oral epithelial ALI culture that appears to fully recapitulate in vivo oral KSHV infection.
Although KSHV is orally transmitted, viral transmission in the oral epithelia is not well understood. Previous studies have shown that KSHV enters cells by actin-mediated micropinocytosis in endothelial cells or clathrin-mediated endocytosis in fibroblasts [32,33]. CD98, integrins α3β1, αVβ3, αVβ5 and EphA2 receptor have been reported as the initial binding partners of KSHV to the cell surface [20,34,35]. Here, we found that the apical surface of intact oral epithelium was resistant to KSHV infection unless we introduced wounding to provide access to the basal layer of oral epithelial tissue. This could be due to a lack of host receptors for KSHV binding or entry at the apical membrane of differentiated tissues since CD98 and Integrin ß are mainly expressed on the basal layer. It is also possible that wounding causes the disruption of physical barrier like tight junction at the superficial layer to allow KSHV infection. Nevertheless, this suggests that KSHV infects the oral epithelia in a manner similar to human papilloma virus [36,37], where infectious virions also require access to basal epithelial cells.
Previous transcriptome analyses of KSHV-infected cells or KSHV-associated tumors have been based on bulk RNAseq study, which disregards cell and tissue heterogeneity [38,39]. However, KSHV infection is a diverse process with uninfected and infected cells along with different stages of viral infection. For example, latent infection and low-level reactivation coexist in KSHV-infected cells and tissues, and both are known to contribute to viral pathogenesis and tumorigenesis. Additionally, the oral epithelium, which is the initial infection site of KSHV, consists of multiple layers of basal, intermediate, and superficial cells. Given that multiple factors contribute to the establishment of KSHV infection, we leveraged the power of scRNAseq to profile gene expression of mock-and KSHV-infected oral epithelial tissues at a single cell level. Despite this strength, scRNAseq also has a number of limitations. The most significant limitation of the drop-seq single cell method is the low sensitivity [40] wherein low abundant viral transcripts are often undetected. For this reason, we used GFP and RFP as surrogate latent and lytic makers, respectively, to differentiate KSHV latent vs. lytic cells and identified the enrichment of latent and lytic cells at the superficial layer of infected tissues at 6dpi. This finding correlates with previous studies that show the presence of lytic cells at the superficial layers [17,18]. Our study also suggests that epithelial cell differentiation signaling and KSHV lytic replication are highly interconnected and potentially affect each other. Furthermore, we found that spontaneous lytic replication resulted in increased virion production over time in 3D oral epithelial tissues, which is also consistent with infectious KSHV particle production and transmission within the oral cavity [7,8,41]. These suggest that epigenetic changes during epithelial differentiation in KSHV-infected 3D epithelial cells may trigger spontaneous viral lytic gene expression, leading to infectious virion production.
KSHV replication not only requires epithelial differentiation, but also leads to the deregulation of epithelial differentiation in 3D oral epithelial ALI culture. Immunohistochemistry of KSHV-infected tissues showed that keratin 5 expression was induced in all layers, whereas S100A7 and keratin 16 expressions were considerably suppressed. scRNAseq also demonstrated that KSHV infection affected epidermal and keratinocyte differentiation, which might be linked with AP-1 activation induced by KSHV-mediated mitogen-activated protein kinase pathway [7,42]. Further sub-clustering of epithelial layers identified the distinctive effects of KSHV on host gene expression within each epithelial layer. For instance, the upregulation of basal layer cell proliferation was likely mediated by KSHV LANA [43,44] and the high lytic reactivation of KSHV at the superficial layer might lead to increased cell death. This suggests that KSHV infection displays distinctive effects on host gene expression in different epithelial layers, ultimately modulating its own viral lifecycle.
KSHV gene expression is used to classify viral lifecycle to latent, immediate early lytic, early lytic, and late lytic infection. To date, it is still unknown how KSHV gene expression is precisely regulated during de novo infection at a single cell level. Interestingly, we found a unique population of GFP+/RFP-/PAN-latently infected cells, designated "latent-2", with specific gene expressions at the K2-K5 locus. The previous studies [30,31] have also shown that similar to latent-2 cells, newly de novo infected cells and latently infected BCBL-1 and JSC-1 cells exhibit the transient lytic gene expressions at the K2-K5 locus in 2D culture. It is possible that these latent-2 cells may be in transition from latency to lytic replication or represent the result of an abortive lytic infection following de novo infection. However, it should be noted that even in transitional state or abortive lytic infection, these cells carry distinctive, not random, profile of viral gene expression. In addition, latent-2 cells showed strikingly different phenotypes of various cellular signaling pathways from latent or lytic cells, including cell migration, ephrin receptor signaling, NRF2 response to oxidative stress, wound healing signaling, and apoptosis. Latent-2 cells also showed increased HIF1α signaling that has been previously implicated as a trigger for lytic reactivation [45]. Further study is warranted to investigate the details of latent-2 cells and their role in KSHV biology.
In summary, our study presents a novel KSHV infection model of 3D oral epithelial organoid ALI culture that allows the spontaneous replication and virion production as seen in oral lesions of KS patients. scRNAseq demonstrates that KSHV infection induces drastic changes of epithelial differentiation, which in turn contributes to viral lytic replication. Finally, the study also uncovers a unique population of infected cells with distinct host and viral gene expression. Thus, our in vitro 3D epithelial ALI culture model should be a valuable tool to further dissect KSHV infection and transmission at a basic science level and to screen for antiviral therapeutics to block KSHV transmission at a public health level.

Virus preparation and titration
KSHV infections were performed with recombinant strain KSHV.219 (rKSHV.219) prepared from induced iSLK.219 cells as described previously [46]. Seventy percent confluent iSLK.219 cells were induced with growth medium containing 1 mM sodium butyrate (Sigma Aldrich Cat. No. 303410-100G) and 1 μg/ml doxycycline (Sigma Aldrich Cat. No. D9891) in the absence of puromycin. After 3 days, the virus-containing culture media was collected and cleared by centrifugation and filtering to remove cells and debris. The virus was concentrated by ultracentrifugation for 3 hours at 24,000 rpm in SW32 rotor (Beckman Coulter). Virus pellets were resuspended in DPBS and stored at -80˚C. Infectious virus amounts were determined and calculated by titration on primary normal human oral keratinocytes gingiva (HOKg)

PLOS PATHOGENS
Single-cell analysis of KSHV infection in 3D ALI culture model (Lifeline Cell Technology SKU: FC-0094) cells by measurement of GFP expression using a FACSCanto flow cytometer (BD Biosciences), assuming that one infectious unit (IU) generated one GFP-positive HOKg. Flow cytometry data were analyzed with the FlowJo software.

De novo KSHV infection
EpiOral (ORL-606) tissues were purchased from MatTek and transferred into 6-well plates containing assay medium (GIN-100-ASY) for 1 hour upon arrival. The tissues are then scored with a blunted 31g insulin syringe (BD Cat. 305196). The level of scoring of EpiOral tissues was verified by microscope. Light scoring was determined by an intact layer of cells at the bottom of the wounding. Deep scoring can be determined by whether the insert membrane was exposed in the wounded area. The tissues are then infected on the apical side with 1 x 10 6 IU of KSHV.219 for 4 hr. Following the infection, the tissues were washed with DPBS twice, returned to an air-liquid interface, and kept in 26 ml of assay medium in 10-cm dish. Tissues are incubated at 37˚C for 3 or 6 days before harvesting while changing the media every 2 days.
For KSHV infection of 2D monolayer cultures, HOKg were purchased from LIFELINE and cultured in keratinocyte medium complete kit (Lifeline Cell Technology SKU: LL-0007). Cells were seeded one day before infection. When cell reached 60 to 70% confluence, 2 X 10 4 IU of KSHV.219 were added to the cells to obtain an infection similar to the 3D culture infection. After incubation for 4 hours at 37˚C, cells were washed with DPBS twice, followed by addition of growth medium before being returned to the incubator.

Histology and immunofluorescence staining of ALI epithelial tissue
In order to determine the location of GFP and RFP expressing cells, the tissues were fixed with 10% formalin at room temperature for 1 hour, washed, and snap-frozen in Tissue-Tek O.C.T. Compound (Sakura Finetek USA Inc. 4583). Cryosections of 15 μm were cut and incubated with Hoechst 33342 and then examined by fluorescence microscope (Keyence BZ-X810). To stain for differentiation markers, 100% methanol was added to ALI sections for permeabilization and quenching GFP and RFP fluorescence for 10 min at 4˚C. After permeabilization, ALI sections were incubated with blocking solution consisting of 10% goat serum and 0.1% (v/v) Triton X100 in PBS for 1 hours and then incubated with primary antibodies diluted in 1% BSA-PBS for 2 hours at room temperature. Appropriate fluorescence-conjugated secondary antibodies from goat were incubated for 1 hour. DNA was stained with Hoechst 33342 and mounted with Fluoromount-G (Thermo Scientific 00-4958-02) mounting media. ALI sections were examined under a Leica SP8 confocal microscope. The primary and secondary antibodies used in this study include Keratin 13 (Abcam; ab16112), Keratin 5 (Abcam; ab64081) Keratin 16 (Abcam; ab76416), S100A7 (Abcam; ab13680), Integrin β1 (Santa Cruz; sc-18887), and CD98 (Santa Cruz; sc-21745).

qRT-PCR of KSHV-infected EpiOral tissues
Total RNA was extracted with Tri reagent (Sigma Cat. T9424). 1 ug of RNA was incubated with DNase I (Sigma Cat. AMPD1) and reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad Cat. 1708891). The resulting cDNA was used for qPCR with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Cat. 1725270). Primer sequences used for qRT-PCR are found in S2 Table. Measuring KSHV genome copy numbers Supernatant collected at indicated time point were treated with DNase to remove any nonencapsidated viral DNA. DNase was inactivated and viral capsid are disrupted by heating samples at 95˚C for 30 minutes. BAC16 bacmid was used for KSHV genomic DNA standard. Twenty nanograms of DNA was used for qPCR with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad 1725271). qPCR assay used KSHV ORF11. Primer sequences are found in S2 Table. Preparation of EpiOral tissues for single-cell RNA sequencing Mock-and KSHV-infected tissues were harvested at 3 days and 6 days post infection. Tissue inserts were removed from the 6-well plate and placed into a new 6-well plate with enough PBS to coat the bottom. The inside of the insert was then washed with PBS to collect the supernatant. The tissues where then carefully separated from the insert membrane and chopped into small pieces in DMEM. The cells are then incubated for 40 minutes in 2 mL of 1 mg/mL collagenase and dispase (Roche Cat. 10269638001) in a shaking incubator set to 37˚C and 250 rpm. The collagenase and dispase are then inactivated by adding 2 mL of DMEM and 10% FBS. The digested cells are then passed through a 0.45 um cell strainer and then washed with cold PBS with 0.04% BSA (ThermoFisher Cat. AM2618). The filtered cells are also passed through a flowmi cell strainer. Cells are counted with Invitrogen Countess II (ThermoFisher Cat. A27977).

Single-cell RNA sequencing
For this study, three separate infections were used: the first two infection studies used the 10X Genomics Single Cell Gene Expression v2 kit and the third infection study used the v3 kit for the generation of single cell RNAseq libraries. Mock-or KSHV-infected tissues were digested into a single cell suspension and then immediately used for single cell RNA sequencing. Sample preparation and library generation were done following the v2 Rev E (CG00052), v3 Rev A (CG000183). After the libraries were generated, library quality was checked on an Agilent Bioanalyzer 2100 and a low read depth run on an Illumina MiSeq V2 flowcell was used to determine the number of reads required for library sequencing. A larger sequencing run with an Illumina NovaSeq 6000 was then used to fully sequence single cell libraries. Sequencing and demultiplexing was handled by the University of Southern California Molecular Genomics Core. Sequencing data is publicly available at NCBI GEO (GSE184257).

Data analysis of single-cell RNA sequencing
Initial processing of the fastq files was done using 10X Genomics Cell Ranger v3.1. A custom reference was made that combined the human hg38 reference genome, KSHV JSC-1 reference genome (GQ994935.1), GFP, and RFP transcripts. The resulting filtered gene matrices from Cell Ranger was used for further analysis with Seurat v3 and R (version 4.1.1). Initial processing of the fastq files was done using 10X Genomics Cell Ranger v3.1. A custom reference was made that combined the human hg38 reference genome, KSHV JSC-1 reference genome (GQ994935.1), GFP, and RFP transcripts. The resulting filtered gene matrices from Cell Ranger was used for further analysis with Seurat v3 and R (version 4.1.1). Cells with a mitochondrial read percentage greater than 10% or expressing less than 200 genes were excluded from analysis (S4 Fig). To account for potential batch effects between the three sets of data, we used the sctransform [47] and glmGamPoi [48] packages to normalize and stabilize variance in the data.
To annotate the epithelial layer of each cell, we chose gene expression markers that have been previously reported [10][11][12][13][14][15][16]. Canonically, there are four layers in the oral epithelia: basal, spinous, granular, and cornified. From our data, we found it difficult to differentiate between the spinous and granular layers, so these two layers were combined and classified as the intermediate layer. Basal cells were identified by enrichment of KRT5, KRT14, and S100A2. Intermediate cells were identified by enrichment of NOTCH3, S100A4, and KRT13, and TGM5. Superficial layer cells were identified by enrichment of S100P, SPRR2A, and SPRR2E. (S4 Fig.).
KSHV-infected cells were identified by expression of GFP transcripts. If cell was GFP+ and PAN+ or RFP+, cell was annotated as a lytic cell. If cell was GFP+, RFP-, and PAN-, cell was categorized as a latent cell. Latent-2 cell was identified as GFP+ cell with K2, K3, or K4 expression.
Differentially expressed genes were identified using the FindMarkers function with a cutoff of p-value < 0.05 and |fold change| > 1.5. Gene ontology was performed on the genes using either the PANTHER Classification System [49,50] or Qiagen's Ingenuity Pathway Analysis (QIAGEN Inc., https://digitalinsights.qiagen.com/IPA) [51]. Bar charts were generated with Prism 9.0.0 (Graphpad Software, LLC). Pathway bubble plots were generated with the ggplot2 package [52] and heatmaps were generated with pheatmap (https://cran.r-project.org/web/ packages/pheatmap/index.html).  Table. Primers used for qPCR assays. Table of forward and reverse primers used for qPCR assays used in Fig 1. (TIFF)