s2fieldsofstudy: Biology Medicine

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Effects of High-Intensity Interval and Moderate-Intensity Continuous Exercise on Inflammatory, Leptin, IgA, and Lipid Peroxidation Responses in Obese Males

Purpose: To compare the effects of a single high-intensity interval exercise session (HIIE) with a moderate-intensity continuous exercise session (MICE) on the inflammatory profile, IgA levels, and lipid peroxidation in sedentary obese males. Methods: Ten sedentary obese men (age 28.5 ± 2.7 years; BMI 35.9 ± 4.9 kg/m2; body fat 40.6 ± 2.0%) performed three experimental sessions, on separate days with 1 week wash-out period between interventions, according to a randomized order: (1) HIIE: 10 × 60 s at 90% of the HRmax alternated by 60 s of active recovery; (2) MICE: 20 min at 70% of the HRmax; (3) Rest—control. Blood and saliva samples were collected before, immediately after and 60 min after the end of each session in order to analyse serum levels of cytokines, IgA, and lipoperoxidation markers. Results: Leptin levels decreased immediately after HIIE (P = 0.033) and was different from the MICE (P = 0.025). IFN-γ levels were reduced immediately after (P = 0.032) and 60 min after HIIE (P = 0.003) compared to baseline, and it also increased IL-4 levels immediately after exercise (P = 0.007) compared to resting values. MICE promoted an increase in IFN-γ levels immediately after exercise (P = 0.025) and 60 min after exercise (P = 0.004) in relation to baseline. Both exercise conditions increased IL-6 levels up to 60 min after exercise (P < 0.05). The IFN-γ/IL-4 ratio decreased immediately after (P = 0.002) and 60 min after HIIE (P = 0.005) in relation to pre-exercise. No changes were found for IgA-S and TBARS for any of the conditions. Conclusion: A single HIIE session is able to decrease IFN-γ/IL-4 ratio, indicating an anti-inflammatory response, without alterations in the function of the mucosal immune system and lipoperoxidation. On the other hand, a brief session of MICE induced changes in the pattern of cytokines associated with increased cellular immune function.

INTRODUCTION
Obesity and chronic low-grade inflammation are important risk factors for the development of cardiometabolic diseases (Grundy et al., 2004;Phillips and Prins, 2008). Adipose tissue is considered a complex endocrine organ capable of regulating the immune system and inflammation through the production of adipokines (Galic et al., 2010;Wronska and Kmiec, 2012). Leptin, for example, is an adipokine capable of modulating the activity of proinflammatory phenotype T helper lymphocytes (Th1) (Apostolopoulos et al., 2016) and sympathetic nervous system (da Silva Rossato et al., 2014;Krause et al., 2015). The increase in leptin production seems to trigger an increase in the infiltration of immune cells into visceral adipose tissue (VAT) in response to obesity (Wensveen et al., 2015). Th1 lymphocytes produce interferon-γ (IFNγ), an important inducer of macrophage polarization to a pro-inflammatory phenotype (M1), and also contribute to the increase of systemic inflammation through the production of inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) (Falcão-Pires et al., 2012;Wensveen et al., 2015).
The production of cytokines by T lymphocytes plays an important role in the adaptive immune response. Th1 (T1) lymphocytes act on the cellular immune response playing a major role in infections, especially against bacteria and viruses. Anti-inflammatory Th2 (T2) phenotype lymphocytes produce interleukin-4 (IL-4) and are responsible for the humoral immune response and prevention of helminth infections and response to allergen agents (Kidd, 2003). The imbalance in the IFN-γ/IL-4 ratio is reported in acute and chronic infections, and it is used as an indicator of the T1/T2 ratio (Zhao et al., 2012). Excess body fat and high levels of pro-inflammatory cytokines are conditions associated with impaired immune systems and greater susceptibility to the development of infectious diseases (Huttunen and Syrjänen, 2012;Kanneganti and Dixit, 2012;Kaspersen et al., 2015Kaspersen et al., , 2016. Moreover, in vitro stimulation with leptin in cells of obese individuals resulted in lower IFNγ production by Natural Killers (NK) cells compared to lean individuals (Laue et al., 2015).
The regular practice of moderate-intensity continuous exercise (MICE) has a positive effect on the inflammatory profile and on the immune system of sedentary obese individuals (Walsh et al., 2011;Trochimiak and Hübner-Wozniak, 2012;Abd El-Kader et al., 2013). Accumulating at least 150 min of MICE per week is recommended for health maintenance in adult subjects (Garber et al., 2011). However, a lack of time is reported as one of the main barriers to the regular practice of physical exercise (Burgess et al., 2017). Thus, high-intensity interval exercise (HIIE) emerges as a time-efficient alternative compared to traditional exercise recommendations as it promotes similar metabolic adaptations to MICE with a lower commitment of weekly time, which may provide greater adherence to the regular practice of physical exercise (Little et al., 2014;Lanzi et al., 2015).
According to previous studies, a single HIIE session is capable of promoting a transient anti-inflammatory state in healthy and overweight individuals, and it can aid in the control of chronic low-grade inflammation (Cabral-Santos et al., 2015;Wadley et al., 2015;Dorneles et al., 2016). Although interleukin-6 (IL-6) is often associated with pro-inflammatory status in individuals with metabolic diseases in resting conditions (Nimmo et al., 2013), its transient increase during and after exercise is recognized as an acute response with metabolic and anti-inflammatory actions, such as inducing an increase in circulating concentrations of anti-inflammatory cytokines IL-4 and IL-10. This action appears to be proportional to the intensity of the effort and the muscle mass involved (Nimmo et al., 2013). In contrast, it is postulated that high-intensity exercise may promote a suppressive effect on the immune response, reducing the secretion rate of salivary immunoglobulin A (IgA-S) and leading to a transient decrease in the IFN-γ/IL-4 ratio, creating an "open window" for infections (Walsh et al., 2011;Zhao et al., 2012). Changes in the immune system are associated with both exercise intensity and duration (Nieman, 1997). As such, a high-intensity lower-volume exercise may promote less impact on the immune function, and it is important to point out that the acute effect of low-HIIE on inflammatory and immunological responses in individuals with obesity is not wellknown.
Therefore, the present study aimed to compare the acute effect of a single HIIE session and a time-matched MICE session on the inflammatory profile (leptin, IFN-γ, IL-4, IL-6, and IFN-γ/IL-4 ratio), IgA-S and lipid peroxidation (TBARS) in obese sedentary males.

Study Design
This study is characterized as a randomized crossover trial. Sample size was calculated by the statistical power (1-β) with mean and standard deviation of a previous study based on IL-6 (Dorneles et al., 2016), considering a power of 80% and an alpha of 5% (G * Power R , version 3.1.9.2; Institute for Experimental Psychology in Dusseldorf, Germany). The study sample consisted of 10 sedentary adult men according to the international physical activity questionnaire (Craig et al., 2003), and obese (body mass index-BMI > 30 m/kg 2 ; body fat > 25%) (Pi-Sunyer, 2000;WHO, 2016) non-smokers who reported body weight stability in the last 6 months. The study protocol was approved by the Human Research Ethics Committee at UFRN (Protocol No. 976.389 CAAE 42441015.5.0000.5568) and was registered on the Brazilian site of clinical trials (ReBec registration number: RBR-462kr6f). All subjects gave written informed consent in accordance with the Declaration of Helsinki.
The subjects carried out four visits to the laboratory. During the first visit, the anthropometric measurements, maximal treadmill velocity (MTV), and maximum heart rate (HR max ) through an incremental treadmill test (RT250, Movement R , Pompeia, Brazil) were collected. Resting blood pressure (BP) was collected according to the Seventh Brazilian Arterial Hypertension Guideline using the oscillometric method (HEM-7200, OMRON, USA). From the second to the fourth visit, the subjects arrived at the laboratory after a night of fasting for approximately 12 h.
Subjects initially received a standard meal consisting of a nutritional supplement (Hipermassprotein, Atlhética R , Brazil) with an energy content of 4.5 kcal/kg of body weight and diluted in 400 ml of water. Blood collection was performed 45 min after ingesting the standard meal. The subjects were then randomly assigned by a draw to one of three experimental conditions with a 7-day interval (washout) between each session: (1) control; (2) MICE; (3) HIIE (Figure 1).

Anthropometric and Body Composition Assessment
The following anthropometric measurements were performed: body mass and height for calculating BMI and nutritional status. Body mass was measured on a digital scale (BC 553, Tanita R , USA) with a maximum capacity of 150 kg and precision of 100 g, with the individual barefoot and wearing light clothes. A portable stadiometer with an accuracy of 1 mm (Personal Caprice Portatil, Sanny R , Brazil) was used to measure height with the individuals in the Frankfurt position.
The formula used to calculate BMI was: [BMI = body mass (kg) * height (m) −2 ]. Double-energy X-ray absorptiometry (DEXA) was also analyzed (GE, Medical Systems, USA), and the participants were instructed to avoid diuretics and caffeinated beverages the day before the evaluation (Albanese et al., 2003).

Maximal Graded Exercise Test
The participants performed a warm-up on a treadmill (RT250, Movement R , Brazil) at a speed of 2.0 km/h for 3 min. Then they started the incremental test at a speed of 3.0 km/h and increments of 1.0 km/h every minute until voluntary exhaustion. The MTV was considered as the highest velocity sustained by a full stage of 1 min . Heart rate (HR) was monitored throughout the test using a HR monitor (RS800CX, Polar R , Finland) and recorded at the end of each minute. The highest HR value observed during the test was considered as the HRmax. Rating of perceived exertion (RPE) was also monitored and recorded at the end of each minute according to the Borg scale 6-20 (Borg, 1982). The end of the test was determined by the presence of at least one of the following criteria: (i) HR ≥ 100% estimated for age; (ii) RPE > 18; or (iii) when participants voluntarily stopped (Howley et al., 1995).

Low-Volume HIIE and MICE Protocols
HIIE was performed in a 1:1 "effort-recovery." The participants performed 10 × 1 min work bouts at 90% of their individual MTV reached on the maximal graded exercise test, interspersed by 1 min of active recovery at 30% of MTV (i.e., slow walking). This HIIE model on a treadmill was previously published by our research group (Costa et al., 2016;Frazão et al., 2016;Matos et al., 2017;Fayh et al., 2018). The MICE session consisted of 20 min at 65-75% of HRpeak performed continuously, which is the exercise intensity recommended as moderate by the American College of Sports Medicine physical activity guidelines (Garber et al., 2011). The participants performed a 3 min warm-up at 4 km/h before both exercise sessions, and after the exercise sessions they performed a 2 min cool-down at the same speed. HR was continuously recorded throughout the exercise sessions (RS800CX, Polar R , Finland). In addition, whole-body RPE was assessed using the RPE 6-20 Borg scale FIGURE 1 | Summary of experimental design. Standardized meal after 12 h fasting; 45-min resting before exercise; 25-min exercise session (control, MICE, and HIIE); 60-min recovery; HR, heart rate; RPE, rating of perceived exertion. (Borg, 1982) during the last 10 s of each minute during both HIIE and MICE. In the control session, the participants remained in a seated position for 25 min and they were allowed to read and use electronic devices such as a tablet, computer, or smartphone.

Blood Sampling and Saliva Analyses
Blood samples were obtained from the antecubital vein and from the tip of the finger with the subject sitting in order to determine the lactate for the exercise characterization. At the initial visit, 3 mL of whole blood was collected after a night of approximately 12 h fasting for the sample characterization. Biochemical markers were determined by colorimetric methods using commercial kits (DOLES R kit, Brazil) specific for each parameter. In the experimental sessions, 10 mL of venous blood were collected into tubes with a separator gel for serum (Vacuette R , Greiner Bio-One, Brazil) immediately before the beginning of the session, immediately after the session, and 60 min after the end of the session. During the 60 min post-exercise period, the participants remained in a seated position. Food and drink intake was not allowed. However, they were allowed to read and use electronic devices as in the control condition. The last blood sample was obtained after this period. The blood was centrifuged for 15 min at 3,600 revolutions per minute. The serum was then separated into 200 microliter aliquots and stored at −80 • C for further analysis. Concentrations of leptin, IFN-γ, IL-4, and IL-6 were analyzed according to commercial ELISA kits (ELISA Read-Set-Go! R , Ebioscience, USA) following the manufacturer's instructions for analysis on an EZ-Reader microplate reader at 450 nm.
Formation of thiobarbituric acid reactive substances (TBARS) during an acid reaction was used as an index for lipoperoxidation. Samples were first centrifuged at 12,000 g at 4 • C for 10 min, then 250 µL of the sample, 10 µL of 4.5 mM butylated Hydroxytoluene (BHT), and 200 µL of 30% trichloroacetic acid (TCA) were added to 1.5 mL eppendorf tubes. These were subsequently placed in a boiling water bath (100 • C) for 15 min, and centrifuged at 15,000 g at room temperature for 2 min. Next, 400 µL of supernatant and 400 µL of 0.23% thiobarbituric acid (TBA) were pipetted into the cryotubes and boiled in a 100 • C water bath for 30 min. The samples were cooled down for 5 min and pipetted in duplicates of 200 µL into the 96-well plate. TBARS was then determined in a microplate reader at 540 nm. The results were expressed as nmol/mL (Draper and Hadley, 1990).
For the determination of lactate, the tip of the individual's finger was sanitized with a 70% alcohol solution and then pierced with a disposable lancet immediately after the end of the exercise. Approximately 25 µL of blood were collected and analyzed on a portable monitor for lactate determination (Accutrend Plus R , Roche, Switzerland).
Saliva samples were also obtained at the pre-, immediately post-, and 60 min post-exercise with the purpose of determining the salivary IgA-S concentrations at each moment. The saliva was collected without stimulation, with the subjects instructed to rinse their mouths with distilled water and to empty their mouth just before collection. The passive drainage method was used for the collection, in which the volunteer slightly tilted their head forward and allowed the saliva to flow into a pre-weighed and sterilized Falcon tube for a period of 5 min. The tubes were weighed again after collection, so that the volume and the saliva flow rate were estimated. The tubes were weighed with a precision of 0.1 mg with the density of the saliva assumed as (1.0 g.mL −1 ), and the samples were frozen at −80 • C for further analysis. The S-IgA levels were analyzed using commercial ELISA kits (IgA Salivary, DRG, USA). The secretion rate of IgA-S (ng/min) represents the total amount of IgA-S present on the mucosal surface per unit of time; for this, the IgA-S concentration (ng/mL) was multiplied by the saliva flow (mL/min).

Statistical Analyses
Data normality was verified through the Shapiro-Wilk test and the homogeneity analysis of the variances through the Levene test. The exercise sessions were compared in relation to the psychophysiological variables through the Student's t-test. Two-way analysis of variance (ANOVA) was used (condition vs. time) with repeated measures to compare the inter-and intra-condition effects for dependent variables (Leptin, IFNγ, IL-4, IL-6, IFN-γ/IL-4 ratio, TBARS, and IgA-S levels). The sphericity hypothesis was verified by the Mauchly test, and the degrees of freedom were corrected by Greenhouse-Geisser estimates when violated. The size of the variance effect was calculated by partial eta-squared (η 2 p ). Additionally, a correlation between the leptin and IFN-γ concentrations was performed for each condition between the different moments through the Pearson correlation. The data were structured and analyzed using the SPSS statistical package (Statistical Package for Social Sciences Chicago, IL, USA) version 20.0 for Windows and G * Power version 3.1.9.2 (Institute for Experimental Psychology in Dusseldorf, Germany). All results are expressed as mean and standard deviation, and the accepted level of significance was P < 0.05.

Subjects and Exercise Characterization
Subject characteristics are described in Table 1. The results of BMI and fat percentage are compatible with the diagnosis of obesity, while resting BP values indicate that subjects were not hypertensive. Taking into account biochemical analyses at fasting, the subjects, apparently, did not present other significant metabolic alterations.
As shown in Table 2, the HIIE session presented higher values of HR and RPE when compared to MICE, taking into account stimulus values (P < 0.05). Lactate levels, shortly after the exercise, were higher for HIIE when compared to MICE (P < 0.05).  60 min post-exercise. An interaction (condition × time) was observed for leptin [F (4.36) = 4.48, P = 0.005, η 2 p = 0.33] with a reduction (P = 0.033) immediately after HIIE compared to the pre-exercise moment, and a difference between the HIIE and MICE conditions immediately after exercise (P = 0.025). An interaction (condition × time) was also observed for IFNγ [F (4.36) = 9.20, P = 0.000, η 2 p = 0.506]. IFN-γ decreased (P = 0.032) immediately after the HIIE and remained lower than after 60 min compared to pre-exercise (P = 0.003). An increase in both post-exercise (P = 0.025) and after 60 min (P = 0.004) was observed for MICE compared to the pre-exercise moment. Regarding the condition effect, a difference was only found for HIIE after 60 min compared to the control session (P = 0.041). An interaction (condition × time) was also observed for IL-4 [F (4.36) = 4.30, P = 0.006, η 2 p = 0.323] and IL-6 [F (4.36) = 3.31, P = 0.021, η 2 p = 0.269]. IL-4 levels increased only for HIIE shortly after the session (P = 0.007). IL-6 levels increased immediately after (P < 0.001) and 60 min after the end of HIIE (P < 0.001) and MICE (P = 0.031) in relation to pre-exercise. A similar behavior was observed for MICE, both immediately (P = 0.031) and 60 min after the exercise (P < 0.001). The value of 60 min after MICE also presented a difference in relation to the post-exercise (P = 0.033).  Figure 3 shows the IFN-γ/IL-4 ratio for the different conditions at before, after and 60 min after exercise moments. An interaction (condition × time) was observed for the IFN-γ/IL-4 ratio [F (4.36) = 5.80, P = 0.001, η 2 p = 0.392], with a reduction immediately after (P = 0.002) and 60 min after (P = 0.005) the HIIE in comparison to the pre-exercise. In contrast, the MICE did not promote significant changes in the IFN-γ/IL-4 ratio (P > 0.05). Table 3 shows the IgA-S concentrations and IgA-S secretion rate for the different conditions at before, after and 60 min after exercise moments. No interaction (condition × time) was observed for any of the markers. There was also no interaction for the TBARS values (P = 0.518).

Blood/Saliva Cytokine Responses to HIIE and Mice
In addition, we found positive correlations through the Pearson's correlation between leptin concentrations and IFN-γ concentrations for exercise conditions at before, after and 60 min after exercise moments. No correlation was observed for these two cytokines in the control condition (Table 4).

DISCUSSION
Herein, we found that (i) HIIE reduced the leptin levels immediately after the exercise and the IFN-γ/IL-4 ratio up to 60 min post exercise; (ii) MICE increased IFN-γ levels up to 60 min post exercise; (iii) both HIIE and MICE increased the IL-6 levels up to 60 min post exercise; and (iv) no changes in IgA and lipid peroxidation levels were observed following HIIE and MICE. To the best of our knowledge, this is the first study to report changes induced by the HIIE in IFN-γ/IL-4 ratio in sedentary obese males.
IFN-γ is a cytokine mainly produced by NK cells, CD4+ T lymphocytes, and Th1 CD8+, which play a critical role in the innate and adaptive immune response against viruses, some types of bacteria, and protozoa (Schoenborn and Wilson, 2007). Thus, suppression in the production of IFN-γ by T cells after exercise could represent reduced immune defenses against viral and bacterial infections. The reduce in IFN-γ by highintensity physical exercise may represent a temporary decrease in T1-mediated immunity (Walsh et al., 2011). As previously reported, an incremental interval ergometer test, consisting of 3 min of exercise at maximum power, alternated with 30 s of passive rest, showed a decrease in the in vitro soluble levels of FIGURE 2 | Inflammatory profile before (Before), immediately after (After) and 1 h after (After 1 H) high intensity interval exercise (HIIE), moderate intensity continuous exercise (MICE), and control (CON) condition. Leptin (A); interferon-γ (B); interleukin-4 (C); interleukin-6 (D). Values are presented as mean ± sd. a Significant difference compared with before exercise (P < 0.05). b Significant difference compared with before exercise (P < 0.05). *Significant difference compared with HIIE (P < 0.05).
INF-γ after stimulation with anti-CD2/anti-CD28 (Smits et al., 1998). Also, the reduced INF-γ levels after strenuous exercise (1.5 h in 5% downhill running at 75% of maximal capacity) was associated with a low peripheral frequency of memory CD8+ T lymphocytes in the peripheral blood, indicating a transient reduction in immunovigilance against intracellular pathogens (Ibfelt et al., 2002).
In the present study, the reduced IFN-γ levels in addition to an increase in IL-4 concentrations immediately after an HIIE session reinforces the hypothesis that T1 and T2 cells exert reciprocal inhibitory actions on each other after an exercise session (Kidd, 2003). The increase of IL-4 may activate B lymphocytes, promoting their proliferation and differentiation into memory and plasma cells. B cells in plasma contribute to the increase in the amount of circulating antibodies, favoring a first line of defense against antigens and pathogens (Kidd, 2003;Walsh et al., 2011). In this sense, a transient increase in IL-4 levels may favor the humoral immune system against the action of extracellular pathogens. Moreover, considering that IL-4 has anti-inflammatory properties, an increase in the concentrations of this cytokine can help in reducing the inflammatory state and stimulate the polarization of macrophages to their M2 antiinflammatory phenotype (Ignacio et al., 2014;Apostolopoulos et al., 2016).
On the other hand, the reduction in the inflammatory capacity of leukocytes due to exercise may impair immune function. The reduction in the IFN-γ/IL-4 ratio observed in the present study immediately after and maintained up to 60 min after an HIIE session may represent an "open window" for infections associated with decreased cellular immune response in obese individuals (Walsh et al., 2011;Zhao et al., 2012). In contrast, no alterations in IgA levels or in the secretion rate were observed (Table 3). To the best of our knowledge, this is the first study describing the acute influence of different types of exercise on IgA levels in sedentary obese individuals. IgA is an immune mucosal marker and it is considered a first barrier for pathogens. This reduction is related to a higher chance of infections in the upper respiratory tract (Walsh et al., 2011), which could hamper subsequent exercise sessions and thus impair the adherence of individuals who initiate a physical fitness program. Therefore, it is important to point out that our findings suggest that HIIE or MICE session does not compromise the mucosal immunity of inactive and highly sedentary individuals with obesity. In addition, TBARS (an indirect marker of oxidative stress), according to our findings did not increase at the 60 min time point for both exercise types. This is in contrast to the findings of Fisher et al. (2011) who found an increase in TBARS after performing an HIIE protocol. However, this difference may be associated with the type of protocol used in their study (Fisher et al., 2011), as they used HIIE maximal protocol with an "all out" bouts, while the present study used a submaximal HIIE protocol.
It is important to highlight how a low-volume MICE session (i.e., 20 min at ∼70% of HRpeak) improved the T1 response pattern up to 60 min after exercise. Considering that obesity is associated with impairments in the cellular immune response against infectious agents (Huttunen and Syrjänen, 2012;Green and Beck, 2017) , MICE appears to improve the immune response mediated by T1 cells in obese men. Moreover, MICE also increased the anti-inflammatory response by a transient increase of IL-6 up to 60 min after exercise. Together, these results may suggest that 20 min of MICE seems to induce positive changes on inflammatory profile and immune responses in highly inactive sedentary males with obesity. Chronic low-grade inflammation is an important risk factor for developing diseases of cardiometabolic etiology (Grundy et al., 2004;Phillips and Prins, 2008). In this sense, both an HIIE session and a MICE session seem to modulate the inflammatory state, promoting a transient increase in concentrations of anti-inflammatory cytokines in obese subjects (Højbjerre et al., 2007;Dorneles et al., 2016) as observed in the present study (i.e., elevation of IL-6 levels); however, an additional anti-inflammatory effect was observed after HIIE as a function of suppressing the inflammatory response pattern of lymphocytes due to the reduction in the INF-γ/IL-4 ratio.
Leptin is largely expressed in adipose tissue and is recognized for its role in regulating appetite and metabolic control (Falcão-Pires et al., 2012). In the immunological context, this adipokine is responsible for activating the pro-inflammatory phenotype through increased glucose uptake by Th1 cells mediated by glucose transporter-1 (GLUT-1) activity. In addition, leptin is a known activator of the sympathetic system, that directly causes activation of the immune cells (da Silva Rossato et al., 2014;Krause et al., 2014Krause et al., , 2015. Thus, leptin can be considered the link between adipose tissue, metabolic activity and functioning of effector T cells (Saucillo et al., 2014;Apostolopoulos et al., 2016). Serum leptin levels are associated with the amount of adipose tissue. In obesity, the excess of leptin is recognized for promoting an increase in the amount of immune cells in visceral adipose tissue (Wensveen et al., 2015). As leptin is responsible for activating Th1 lymphocytes (Apostolopoulos et al., 2016), its transient reduction can modulate T cell function during exercise. Interestingly, we observed a correlation between the levels of IFN-γ and leptin levels in exercise conditions for the first time (Table 4), which suggests that adipose tissue, along with sympathetic nervous system, may be able to modulate the cytokine pattern secreted by T cells during exercise.
It is important to emphasize that our study was not able to indicate the expression pattern or the cellular origin of these cytokines, since both T and NK cells synthesize and secrete IFNγ into the circulation. It should also be pointed out that the inflammatory and immune response can continue 60 min after the end of the exercise. The follow-up in the present study was only performed up to this moment.
Several studies have suggested HIIE as a time-efficient alternative compared to traditional MICE recommendations (Gibala et al., 2012). This type of exercise is able to increase VO 2max (Lanzi et al., 2015) and improve risk factors for diseases of metabolic etiology in overweight individuals (Gillen et al., 2013;Little et al., 2014). Thus, this study contributes to narrow the gap in the literature on the effects of a HIIE and MICE on inflammatory markers in obese individuals who do not yet present other metabolic alterations, thus collaborating to optimize the exercise prescription for this population. However, further clarification is needed regarding the clinical relevance of the acute effect of HIIE and MICE on the IFN-γ/IL-4 ratio.
In conclusion, the change in the IFN-γ/IL-4 ratio observed after an HIIE session may indicate an anti-inflammatory response, however without compromising the mucosal immune system or increasing oxidative stress. On the other hand, a brief MICE session induced changes in cytokine pattern associated with improved cellular immune response in obese males.


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Atopic dermatitis as Th2 disease revisited

T helper 1 (Th1) and T helper 2 (Th2) populations are a classical dichotomy to understand the immunological disorders, and this concept remains vital in atopic dermatitis (AD). While IL‐4 and IL‐13 are produced mainly by Th2 cells, mast cells, and basophils, these cytokines target these producers, indicating the presence of autocrine and paracrine stimulation. There are two types of receptors for IL‐4 and IL‐13, type I and type II. IL‐4 binds to both type I and II receptors, while IL‐13 has affinity to type II. Dupilumab is an IL‐4Rα‐antagonist that inhibits IL‐4 and IL‐13 signaling through blockade of the shared IL‐4α subunit. The Th2‐stimulating effect of thymic stromal lymphopoietin (TSLP) is triggered by its binding to TSLP receptor (TSLPR) on Th2 cells. The frequency of TSLPR+CD4+ T cells correlates with disease activity. CD4+ T cells directly interact with TSLP to produce a high amount of IL‐4. Conversely, IL‐4 induces TSLPR expression in T cells. Moreover, TSLP and IL‐4 promote CCR4 expression, which is a chemokine receptor for CCL17/TARC. Langerhans cells initiate epicutaneous sensitization with protein antigens and induce Th2 cell–mediated immune responses via TSLP signaling. Filaggrin deficiency, which cornified layer‐damaged skin allows protein antigens to penetrate through the stratum corneum, leads to allergy where IL‐4 and IL‐13 are overproduced. Subsequently, IL‐4 and IL‐13 further depress filaggrin expression with vicious cycle. AD is generally a Th2 disease, but there exist several exceptional and undeniable facts to be considered, including Th1‐activated intrinsic AD, Th1‐infiltrating chronic AD skin lesions, and Th17 involvement.

| INTRODUC TI ON
Atopic dermatitis (AD) is a chronic-intermittent, eczematous dermatitis that starts at infancy or early childhood and persists for a large part of life. A large number of clinical, laboratory, and experimental studies have been performed, but the pathophysiology of AD remains to be clarified.
Th1 and Th2 populations are a classical dichotomy to understand the immunological disorders, 1 and this concept remains vital in clinical and basic research of allergic diseases. When we see the diagnostic criteria of AD by Hanifin and Rejka, 2 we can realize that there are a considerable number of manifestations presumably associated with Th2 cells, including xerosis, ichthyosis, palmar hyperlinearity, immediate skin test reaction, elevated serum immunoglobulin E (IgE), tendency toward cutaneous infection, and impaired cell-mediated immunity. In addition, we have indicated that chondrodermatitis of the auricle 3  Recent use of anti-Th2 cytokine or receptor antibody therapies has been providing important information on Th2-skewing condition of AD. The clinical efficacies of these treatments are mandatory for evaluation of Th2 cytokines in AD pathogenesis. In this review, we revisit the essential role of Th2 cells for AD, but also several issues that are as yet unelucidated merely with Th2 dogma.

| PERTURBATI ON OF S TR AT U M CORNEUM BARRIER IN AD
The barrier function is usually assessed by transepidermal water loss (TEWL) and skin surface hydration. AD patients, especially common, serum IgE-high, extrinsic AD patients, have increased TEWL and lower skin surface hydration, compared with healthy subjects. Even at the non-lesional forearm and lower leg of patients and normal volunteers, the level of skin surface hydration was significantly lower in AD than in normal subjects. 5 The identification of loss-of-function mutations in filaggrin gene (FLG) has shed new light on the mechanisms of AD. 6 When we examined the amount of filaggrin in stratum corneum of AD patients, ichthyosis vulgaris patients, and normal subjects, we confirmed that filaggrin is apparently reduced in ichthyosis vulgaris and AD compared with normal subjects. 7 Patients with FLG mutations have various extents of ichthyosis vulgaris (Figure 1), palmar hyperlinearity ( Figure 2), and atopic dermatitis. Different FLG mutations were F I G U R E 1 Vicious cycle between barrier impairment and allergic disorder in atopic dermatitis. Filaggrin deficiency is a typical cause of barrier impairment (as seen in ichthyosis vulgaris), which allows protein antigens to penetrate through stratum corneum, leading to Langerhans cell-mediated Th2 allergic responses. The resultant upregulation of IL-4/IL-13 production reduces filaggrin expression, resulting in further reduction in barrier and promotion of allergic reaction F I G U R E 2 Palmar hyperlinearity associated with FLG mutations. The intensity of hyperlinearity depends on the age of patients reported depending on races, and the frequency of FLG mutations is 24.3% in AD patients in Hamamatsu University Hospital. Profilaggrin is the major component of the keratohyalin granules within epidermal granular cells. During epidermal terminal differentiation, the profilaggrin polyprotein is dephosphorylated and rapidly cleaved by serine proteases, such as kallikrein-5, 8 to form monomeric filaggrin, which is further degraded into natural moisturizing factors.
Perturbation of skin barrier function as a result of reduction or complete loss of filaggrin expression enhances percutaneous transfer of allergens ( Figure 1). The association of the FLG mutations in particular with the extrinsic type of AD was observed. 9 Thus, the skin barrier is perturbed in AD, and FLG mutation represents a typical cause of barrier impairment. The allergic conditions of AD may be preceded by skin barrier impairment, which allows protein antigens to penetrate through disrupted barrier.

| TH2-P OL ARIZED IMMUNOLOG IC AL S TATE OF AD
In general, skin responses to exogenous antigens can be divided into three types, depending on the evoked timing after exposure, the eruption type, and the immunocompetent participants ( Figure 3).
The responses comprise immediate-, late-phase, and delayed-type reactions. AD is well known as a Th2-polarized disease. Th2 cells are involved mainly in the late-phase reaction, 10 but the Th2 reaction may extend to parts of the immediate-and delayed-type hypersensitivity, because of graduation between these three categories. In the chronic lesion of AD, Th1 cells (additionally Th22 and Tc1 cells) are considered to induce chronic eczema with thick epidermis.
As expected with elevated total serum IgE and circulating eosinophils, AD patients show high levels of Th2 cytokines, such as IL-4, IL-5, and IL-13. 11 Along with the elevation of IL-5, eosinophil counts and eosinophil cationic protein levels are increased in AD. 12 In addition to AD, there are many Th2-polarized skin diseases such as eosinophilic pustular folliculitis, prurigo, angiolymphoid hyperplasia with eosinophilia (Kimura's disease), eosinophilic cellulitis (Wells' syndrome), bullous pemphigoid, and cutaneous T-cell lymphoma.
Among them, AD is a disorder where the epidermis and dermis afford Th2-associated inflammatory sites.
To identify Th2 cells, several methods are used. Intracellular cytokine staining and subsequent flow cytometry are a standard method, and IL-4-or IL-5-positive CD4 + cells represent Th2 cells.
The surface marker of CD4 + CD7 − CCR4 + phenotype is also employed. Chemotaxis toward CCL17/TARC represents functional identification. 13  In this sense, it is an issue which cell type is the initial producer of IL-4. One candidate for this "early IL-4" producer is basophil. It is noted that IL-33 stimulates basophils to secrete IL-4. 16 There are two types of receptors for IL-4 and IL-13 ( Figure 4).

| IL-4/IL-13 AND THEIR RECEP TOR S
Both receptors are comprised of heterodimers. Type I consists of IL-4Rα and Common γ, and type II is composed of IL-4Rα and IL-13Rα1.
IL-13Rα2 is another IL-13 receptor, serving as either a decoy or a key mediator of fibrosis. IL-4 binds to both type I and II receptors, while IL-13 has affinity to type II and IL-13Rα2. Dupilumab is an IL-4 Rαantagonist that inhibits IL-4 and IL-13 signaling through blockade of the shared IL-4α subunit. 17 In addition to allergy, IL-4/IL-13 regulates the immune microenvironment in cancer. Signaling via type II receptor induces tumor proliferation, cell survival, cell adhesion, and metastasis. 18 Thus, type II expression in tumors is associated with poor prognosis.

| PROMOTI ON OF IL-4 S TIMUL ATI ON/ PRODUC TION BY AD -AGG R AVATING FAC TOR THYMIC S TROMAL LYMPHOP OIE TIN IN TH2 CELL S
CD4 + CCR4 + CXCR3 − CCR7 − CCR10 + CLA + Th2 cells in AD patients exhibit enhanced TSLPR expression. 20 The frequency of TSLPR + CD4 + T cells correlates with disease activity. CD4 + T cells directly interact with TSLP to produce a high amount of IL-4.

Conversely, IL-4 induces TSLPR expression in T cells. Moreover,
TSLP and IL-4 promote CCR4 expression, which is a chemokine receptor for CCL17/TARC. Thus, the scheme shows mutual activation of IL-4 production, TSLP receptor expression, and CCR4 expression by IL-4 and TSLP in Th2 cells.
Likewise, many AD-aggravating factors directly or indirectly induce stimulation of Th2 cells and resultant IL-4 production ( Figure 6).
Protein antigens induce Th2 responses, while metals and haptens usually evoke Th1 responses. 21 In the sweat, Malassezia antigen may deteriorate Th2 aspect of AD, 22 while nickel, cobalt, and chromium are usually Th1 inducers. 9 Mental stress or anxiety is associated with Th2 status. 23 In addition, future studies on microbiota might shed light for its association with Th2 condition. 24

| REL ATIONS HIP B E T WEEN BARRIER S TATUS AND TH2 RE S P ON S E S IN AD
The skin immune status is closely associated with the disordered condition of skin barrier. 28 Barrier disruption induces Th2 cytokine production and eosinophil infiltration in mice. 25 Topical protein antigen application further promotes Th2 responses. 29 Therefore, barrier impairment results in elevation of Th2 status.
Filaggrin deficiency, which cornified layer-damaged skin allows protein antigens to penetrate through the stratum corneum, leads to allergy where IL-4 and IL-13 are overproduced. Subsequently, IL-4 and IL-13 further depress filaggrin expression. 30 This cascade yields vicious cycle, and therefore, even heterozygous FLG mutations may result in development of AD. In addition, IL-4 suppresses the enhancement of ceramide synthesis and cutaneous permeability barrier functions, which further aggravates the barrier. 31 Neutralization of the normally acidic stratum corneum has deleterious consequences for permeability barrier homeostasis and stratum corneum integrity/cohesion attributable to serine proteases activation, leading to deactivation/degradation of lipid-processing enzymes and corneodesmosomes. 32

| L ACK OF PURIT Y OF TH2 P OL ARIZ ATI ON IN AD
Although several criteria for its definition have been widely approved, there still exist variations in the diagnosis of AD because of its heterogeneous aspects. AD is generally a Th2 disease, but there exist several exceptional and undeniable facts to be considered.

| Intrinsic AD and Th1 activation
The first issue is the presence of intrinsic type among AD patients.
AD can be categorized into the IgE-high, extrinsic type and the IgEnormal, intrinsic type. 21 While extrinsic AD is the classical type with high prevalence, the incidence of intrinsic AD is approximately 20% with female predominance. The skin barrier is perturbed in the extrinsic, and FLG mutation represents a typical cause of barrier impairment. Intrinsic AD is immunologically characterized by the higher expression of IFNγ and non-protein antigens, such as metals and haptens, may induce dermatitis. 9,21 The overproduction of IFNγ may further downregulate IgE production in intrinsic AD, as suggested by our in vitro study, 9 and intrinsic AD is linked with much lower levels of IL-4 and IL-13. 33

| Chronic AD lesion and Th1 infiltration
It is known that Th2 cells infiltrate in the acute lesion of AD, but Th1 cells, and presumably Tc1 cells, are involved in the chronic lesion.
The acute skin lesion corresponds to the late-phase reaction evoked by Th2 cells and eosinophils, while the chronic skin lesion corresponds to the delayed-type hypersensitivity induced by Th1/Tc1 cells. 34 Accordingly, IFNγ generally plays an essential role for occurrence of dermatitis. 26 Clinically, the therapeutic effectiveness of systemic IFNγ, which suppresses Th2 cells in vitro, is limited, further supporting the notion that AD does not have a pure Th2 property.

| Involvement of Th17 cells
Th17 cells, producing IL-17A and IL-22, are increased in the peripheral blood of AD, and Th17 cells infiltrate in the acute skin lesion F I G U R E 6 Causative and aggravating factors in atopic dermatitis more markedly than in the chronic lesion. 35 There is a tendency that the frequency of circulating Th17 cells is higher in intrinsic AD than in extrinsic AD. 9 In the lesional skin, another group of investigators found positive correlations between Th17-related molecules and SCORAD scores in patients with intrinsic AD, whereas only patients with extrinsic AD show positive correlations between SCORAD scores and Th2 cytokine (IL-4 and IL-5) levels. 36 Asian patients may be more prone to have Th17-participating AD. 37

| THER APIES FOR IMMUNOMODUL ATION OF AD
Recent cytokine-targeting biologic therapies have been proving the disease mechanisms underlying atopic dermatitis as well as psoriasis.
The essential involvement of IL-4 and IL-13 in AD pathogenesis is strongly supported by the therapeutic effectiveness of anti-IL-4Rα antibody dupilumab, which block IL-4 binding to type I and type II IL-4 receptors and IL-13 binding to type II receptor ( Figure 4).
Dupilumab depresses Th2-centered inflammatory axis 38 and exerts a high clinical efficacy. Dupilumab was approved in Japan in 2018, and the number of patients treated with this biologic has been increasing. Before dupilumab was marketed, many patients with refractory AD had been treated with cyclosporine A. However, because of its renal toxicity, careful attention is necessary for its long term use.
Dupilumab is beneficial for such cases.
There are possible Th2 cytokine-targeting therapies, including anti-IL-13 antibody 39 and anti-IL-31 antibody. 40 Furthermore, semi-Th2 targeting JAK inhibitors are promising therapeutic modalities for AD. Further clarification of the immunological mechanism of AD may lead to development of novel therapies. 19

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.


extfieldsofstudy: Biology Chemistry

text:
Identification of substrates for the conserved prolyl hydroxylase Ofd1 using quantitative proteomics in fission yeast

Prolyl hydroxylation functions in diverse cellular pathways, such as collagen biogenesis, oxygen sensing, and translation termination. Prolyl hydroxylation is catalyzed by 2-oxoglutarate (2-OG) oxygenases. The fission yeast 2-OG oxygenase Ofd1 dihydroxylates the 40S ribosomal protein Rps23 and regulates the hypoxic response by controlling activity and stability of the sterol regulatory element-binding protein Sre1. Multiple substrates have been found for 2-OG oxygenases, yet the only known substrate of Ofd1 and its homologs is Rps23. Here, we report the first fission yeast prolyl hydroxylome and demonstrate that hydroxylation is more prevalent than previously known. Using quantitative mass spectrometry, we identify Rpb10, a shared subunit in RNA polymerase I, II, and III, as a novel Ofd1 substrate. In addition, we discovered six Ofd1 binding partners and 16 additional Ofd1 candidate substrates. Although Ofd1 promotes Sre1 degradation, proteomic analysis revealed that Ofd1 does not broadly regulate protein degradation. Instead, the effect of Ofd1 on the proteome is through negative regulation of Sre1N. Finally, we show that the interaction between Ofd1 and the Sre1 bHLH region is conserved across Sre1 homologs suggesting that Ofd1-dependent regulation of SREBPs may be conserved in other fungi. Collectively, these studies provide a new dataset of post-translational modifications and expand the biological functions for a conserved prolyl hydroxylase.

Introduction 30
Eukaryotes require environmental oxygen for essential metabolic processes. Cells exist in

85
Yeast strains were grown to exponential phase at 30°C in yeast extract plus supplements 86 (225 µg/ml each of histidine, leucine, adenine, lysine and uracil) or in SILAC medium 87 (Edinburgh minimal medium plus 75 µg/ml each of histidine, adenine, leucine and uracil and 30 88 µg/ml of regular or heavy lysine) [29].

90
Yeast two-hybrid screen 91 Pairwise yeast two-hybrid assays were performed following the Clontech Matchmaker™ GAL4 92 Two-Hybrid System 3 user manual, using the yeast two-hybrid S. cerevisiae strain AH109 [30].
162 filtered at a 1% FDR. Dynamic modification was chosen for carbamidomethyl C, oxidation M, 163 deamidation of N and Q, hydroxylation of P, dihydroxylation of P, TMT 6-plex for N-term and 164 K.
165 Data processing 166 The protein area calculated by Proteome Discoverer Software was used to quantify protein 167 abundance. The relative abundance of each protein in each of the 10 TMT channels was 168 calculated as following: 170 The relative abundance of WT5 and ofd1Δ2 samples were higher than the other replicates and 171 therefore these two samples were excluded from quantile normalization and further analysis. The 172 remaining eight samples were quantile normalized using 'preprocessCore' package embedded in 173 R [32]. The normalized relative abundance was saved and used for further analysis. Student's t-174 test was applied to calculate the p-value of protein relative abundance in each sample using R.

175
At the peptide level, the precursor area was used to quantify PSM abundance. The 176 relative abundance of each PSM in each of the 8 channels was calculated as following: 242 288 peptides. One peptide from Tif32 (eIF3a) contained a monohydroxylated proline residue with 289 0.340 heavy to light ratio (wild-type/ofd1Δ cells), indicating that the modification is Ofd1-290 independent (Fig S3). Thus, while the yeast two-hybrid screen indicates that Ofd1 binds to eIF3 291 subunits, we did not find evidence that eIF3 subunits are Ofd1 substrates.

292
In addition to subunits of the eIF3m complex, we identified 1263 proteins in the eIF3m

298
We defined prolyl hydroxylated peptides with a heavy to light (H/L) ratio > 3.5 as Ofd1-299 dependent. Using this criterion, two unique prolyl dihydroxylated peptides from Rps23 300 (IGVEAKQP(OH) 2 NSAIRK and IGVEAKQ(deamidated)P(OH) 2 NSAIRK) had H/L ratios of 301 14.941 and 20.030 respectively, confirming that Rps23 P62 dihydroxylation is Ofd1-dependent 302 and validating the dataset. To expand our search for Ofd1 substrates, we repeated the analysis 303 using a FDR of 0.1 and plotted the prolyl hydroxylated peptides based on their H/L and 304 precursor area ( Fig 2D). Again, we defined Ofd1-dependent hydroxylated peptides as having a 305 H/L ratio > 3.5 ( Fig 2D). After manual inspection of the spectra, we identified 16 novel Ofd1 306 candidate substrates, including 9 monohydroxylated peptides, 4 dihydroxylated peptides, and 3 307 peptides containing one mono and one dihydroxylated proline (Table 1, Fig S2). The prolyl 308 dihydroxylated peptide from Nup124, for instance, had complete y-ion series around the proline 309 residue and was more abundant in WT (heavy) cells, indicating the hydroxylation was Ofd1-310 dependent (Fig 2E). For PSMs with incomplete b-or y-ion series, we made specific assumptions 311 about other residue modifications in order to classify these PSMs as prolyl hydroxylated ( 339 Indeed, in WT cells (heavy) > 99% of the peptides from Rps23 were dihydroxylated, whereas in 340 ofd1Δ cells (light) 92% were non-hydroxylated ( Fig S1C). The non-hydroxylated peptides for 341 Fas1 and Rrp5 did not dramatically increase in ofd1Δ cells, perhaps due to the fact that the 342 hydrophobic non-hydroxylated peptides were easier to detect. However, the non-hydroxylated 343 peptide from Rpb10 increased from 52% in WT cells to 96% in ofd1Δ cells, indicating that 344 monohydroxylation of Rpb10 is Ofd1-dependent ( Fig S1C). Together, these data indicate that 345 Rpb10 is an Ofd1 substrate that is present in mono-hydroxylated and non-hydroxylated forms.
346 Additional experiments are required to quantify accurately the two Rpb10 populations. Analysis of the complete eIF3m prep also revealed Ofd1-independent prolyl 348 hydroxylation (H/L ≤ 3.5). Using a strict FDR of 0.01, we discovered 7 prolyl hydroxylated 349 peptides with complete b-or y-ion series around the modified proline residue (

364
In the eIF3m dataset, we observed that a fraction of prolyl hydroxylated peptides (20.9%) 365 deviated from the center with H/L ratios between 0.330 and 0.660 (Fig 2D). This phenomenon 366 was also observed in the entire collection of peptides identified in the eIF3m prep (Fig S4A), 367 indicating that it was independent of prolyl hydroxylation. When H/L ratios were calculated for 368 eIF3m interacting proteins rather than peptides, the left-shifted population disappeared indicating 369 that the phenomenon was likely due to result of noise at PSM level rather than from biological 370 differences between WT and ofd1Δ cells (Fig S4B). In addition, the half-life of proteins 371 containing peptides from the left-shifted population was identical to that of the entire eIF3m prep 372 (Fig S4C), indicating the shift was not due to differences in protein turnover [42]. 379 each strain were used for quantile normalization and further analysis (Fig S5). Overall, 2862 380 proteins were detected, which is comparable to published fission yeast proteomics results (Fig   381 3A) [42]. We searched for prolyl hydroxylation as described previously using a FDR of 0.01 and 382 identified 38 peptides with prolyl dihydroxylation, 122 peptides with prolyl monohydroxylation, 383 and 3 peptides with both mono and dihydroxylation. These PSMs represented a small fraction of 384 total PSM detected (0.2%) and mapped to 128 proteins. After manual inspection, we identified 385 12 peptides with complete b-or y-ion series around the modified proline residue ( 432 (CNJ02310) in C. neoformans (Fig 4A, 4B). Hms1 and Tye7 are SREBP-N proteins that lack the 433 transmembrane segments, suggesting that Ofd1 can regulate SREBP-N proteins in addition to 434 proteolytic product of SREBP [9]. Each of these organisms contains an Ofd1 homolog with To explore whether Ofd1 regulation of Sre1N exists in other fungal organisms, we tested 504 whether this binding interaction is conserved. Yeast two-hybrid analysis between Ofd1 and the 505 bHLH domains of fungal SREBP or SREBP-N proteins showed that four SREBP and SREBP-N 506 proteins from S. cerevisiae, C. albicans, and C. neoformans interacted with Ofd1, suggesting a 507 conserved regulation of SREBPs by Ofd1 in these species (Fig 4). We recently identified an 508

515
Prolyl hydroxylation plays different roles in cells, ranging from a structural function in 516 collagen biogenesis to regulation of the hypoxic response. In yeast, Rps23 is the only known 517 prolyl hydroxylated protein, and hydroxylation functions to regulate Sre1N and promotes 518 translational fidelity [17,18]. In this study, we performed the first search for prolyl 519 hydroxylation in fission yeast, and we discovered 12 additional prolyl hydroxylated proteins with 520 complete mass spectrometry fragmentation around the hydroxylated proline. Even without an 521 enrichment method, 4.2% of proteins were prolyl hydroxylated in our proteomic search, 522 suggesting that prolyl hydroxylation is more prevalent than previously known. The development 523 of a robust enrichment method is critical to fully reveal the prolyl hydroxylome.

524
The 530 Together, these findings suggest that the ribosome is a common target for protein hydroxylation.
531 Further characterization of these modifications and their corresponding oxygenases will provide 532 new insights into translation and cell proliferation.


extfieldsofstudy:
Medicine
Materials Science

s2fieldsofstudy: Biology Physics

text:
The voltage-dependence of MscL has dipolar and dielectric contributions and is governed by local intramembrane electric field

Channels without canonical voltage sensors can be modulated by voltage acting on other domains. Here we show that besides protein dipoles, pore hydration can be affected by electric fields. In patches, both WT MscL and its V23T mutant show a decrease in the tension midpoint with hyperpolarization. The mutant exhibits a stronger parabolic dependence of transition energy on voltage, highly consistent with the favourable dielectric contribution from water filling the expanding pore. Purified V23T MscL in DPhPC droplet interface bilayers shows a similar voltage dependence. When reconstituted in an asymmetric DOPhPC/DPhPC bilayer carrying a permanent bias of ~130 mV due to a dipole potential difference between the interfaces, the channel behaved as if the local intramembrane electric field sets the tension threshold for gating rather than just the externally applied voltage. The data emphasize the roles of polarized water in the pore and interfacial lipid dipoles in channel gating thermodynamics.

from the position of its in-vivo function as a bacterial 'safety valve' , as well as from the point of view of bio-inspired material engineering.
The high activation threshold of WT E. coli MscL precluded its studies in artificial solvent-containing membranes (BLM or droplet interface bilayers (DIBs) 17 ), and for this reason the V23T mutant, which is activated at 6-8 mN/m, was chosen for reconstitutions 15 . The previously observed higher probability of opening events in the course of cyclic mechanical stimulation of the DIB-reconstituted V23T MscL at membrane voltages above 80 mV 15 motivated us to look more carefully at the voltage dependence of WT and V23T MscL. Here, we present patch-clamp measurements obtained under linear pressure ramp stimulation at different voltages and evaluate voltage contributions to the opening transition energy of these two channel variants. The data reveal that both versions of MscL have dipole (linear with voltage) and dielectric/capacitive (quadratic with voltage) components that contribute to the transition energy. In contrast to WT, which exhibits a larger dipole and a smaller dielectric contribution, V23T shows a dominating dielectric effect likely related to the more pronounced hydration of the mutant pore 3,4 . The increasing aqueous volume of the pore and the flattening of the protein complex impart the capacitive/dielectric energy contribution that stabilizes the expanded and subconductive states of the mutant channel. To further explore the nature of the electric field effect that influences the open probability, we have compared the V23T MscL behaviour in electrically symmetric and asymmetric DIBs where a large intrinsic electric bias was created by using lipids with different interfacial dipole potentials. The data obtained in DIBs confirm that V23T MscL senses the local intramembrane electric field in the membrane core between the interfacial layers, and thus the voltage imposed by external electrodes is sensed not directly, but rather in combination with the potential imparted by the lipids.

Results
MscL exhibits dipole and dielectric components of voltage dependence in patch-clamp experiments. Figure 1 depicts activation curves of WT and V23T MscL populations recorded using the standard patch-clamp technique in inside-out patches excised from giant spheroplasts (~30-50 channels/patch). The mechanical stimulus is a linear ramp of pressure difference between the pipette and the bulk from zero to saturating level (~250 mm Hg), repeated at different maintained voltages. We should explain that this pressure gradient produces tension in the curved patch membrane according to the law of Laplace. This tension is the primary stimulus driving channel opening. One can see that in wild-type MscL (Fig. 1a) the midpoint for population activation does not change significantly at negative pipette voltages (asterisk) but shifts to the left with positive . Experiments were conducted in inside-out patches excised from giant bacterial spheroplasts expressing corresponding channels. Pressure midpoints for WT MscL (a) are higher at negative pipette voltages (asterisks), whereas V23T MscL (b) shows shallower and more symmetric responses and a more substantial shift to the left of the pressure midpoint with voltage. Both channels show 'lingering' conductive states at high negative voltages after stimulating pressure is released (black arrows). Plots of the closed-to-open transition energy extracted from activation curves as a function of pipette voltage (c,d). Parabolic fits predict different contributions from the capacitive (quadratic) and dipole (linear) components of the energy dependence on membrane potential, V (represented by right axes on panels c and d). This behaviour was reproduced in six independent patches for WT MscL and nine patches for V23T.
voltage. For V23T MscL, in contrast, the midpoint shifts considerably to the left under both voltages. The clear difference is in the slope of activation curves. For WT MscL, the curves are steeper, which correlates with higher cooperativity of gating (predominant all-or-nothing transition between the closed and fully open states), whereas the lower slope of V23T curves is ascribed to high occupancy of subconductive states at low tensions and pre-expanded closed state (i.e. smaller expansion area resulting in shallower dependence on tension) 3 . The relationships between population current are presented in Methods. We note that the tension midpoints at low voltages for WT and V23T MscL reside at 12 and 8.3 mN/m, respectively, based on previous experimental studies 3,6 . Assuming that the patch geometry (curvature) in all trials at different voltages within any particular experiment remains the same, reduction of midpoint tension γ 0.5 can be interpreted as a reduction of the effective energy for the closedto-open transition. We observed an unusual common feature: both channels show 'lingering' low-conductance states at high negative voltages after stimulating pressure is released (black arrows).
To interpret the shifts in activation curves, we assume equilibrium conditions at any tension and use the Boltzmann-type relationship for the closed and open state probabilities (P o and P c ).
E o is the free energy gap between the closed and open states in the absence of tension, γ is membrane tension, ΔA is the protein area change in the plane of the membrane, k is the Boltzmann constant, and T is temperature. From this relationship, one can see that at the midpoint (γ 0.5 ) tension, where P o is equal to P c , the exponential term is zero, which sets the transition energy in the absence of tension E 0 = γ 0.5* ΔA. The fact that γ 0.5 decreases with voltage indicates that there must be voltage-dependent terms in the transition energy. Knowing midpoint tensions at low voltages and transition-associated area changes, we calculated the transition energy at each voltage. In our previous studies 3, 6 , the E o and ΔA parameters were estimated as 58 kT and 20 nm 2 for WT MscL and E o = 41 kT and ΔA = 18 nm 2 for V23T MscL, respectively. As seen from Fig. 1c, the energy for WT MscL slightly increases at negative pipette voltage and decreases at positive pipette voltages. For V23T (Fig. 1d), the voltage-dependent term is always negative at both voltages (more pronounced at positive potential) and the E o (V) dependence can be fitted with a parabola with a dominating negative quadratic term. The parabolic fit of the WT dependence indicates a larger linear term (Fig. 1c). The linear and quadratic terms can be physically interpreted as the dipolar (E d ) and dielectric (capacitive, E c ) contributions of the electric polarization of the system and thus the transition energy in the absence of tension can be written as.
where E i is the voltage-independent term estimated at low voltages. The open probability, therefore can be written in the form: Conceptually, the constant K d can be interpreted as an effective charge crossing the voltage V, while K c as an effective capacitance that, being charged, gains energy proportional to V 2 . However, given that the effective dipole of the channel MscL changes during the gating transition and crosses a varying fraction of the transmembrane voltage, and also considering that the capacitance of the channel/lipid/water system is defined by heterogeneous parts with a changing geometry, interpretation of the coefficients as "fixed" effective parameters might be an oversimplification. The combination of parameters contributing to these changing coefficients is provided in the Supplement for our cylindrical multilayer model system. The shape of parabolic fitting curves of the total transition energy and fitting parameters indicate that the capacitive (dielectric) term described by the quadratic dependence on voltage dominates in V23T MscL and the dipolar (linear) term in the mutant is diminished compared to WT. Estimations based on the parabolic fits (Fig. 1c,d) show that at −100 mV pipette potential the unfavourable (opposing opening) dipole contributions constitute 1.93kT and 0.53 kT and the favourable capacitive contributions are −1.35kT and −3.11 kT for WT and V23T MscL, respectively. Given that the V23T substitution does not alter the net charge in the pentameric protein but changes the character of pore lining, these disparate electrostatic contributions raise the following questions: what is the nature of the dipole that dominates the WT MscL voltage response and how does the neutral V23T mutation diminish this dipolar contribution? Can the increased hydration of the V23T pore account for the large dielectric contribution in the mutant and where does the localized electric field exert its action in the system? Does the channel feel mostly the applied voltage between the bulk solutions or is it sensitive to the potential in a narrower spatial region where the electrostatic contribution from the membrane interfacial layers is present?
V23T MscL exhibits voltage sensitivity dominated by the dielectric effect in droplet interface bilayers. We explored voltage-sensitivity of V23T MscL in DIBs, which allow us to form membranes with symmetric or asymmetric monolayer lipid compositions characterized with different interfacial dipole potentials 14 . Varying the local electric field inside, between the membrane's boundary layers, would enable us to address the question related to the region where the electric field acts. In the first series of experiments we reconstituted V23T MscL into symmetric DIBs formed of DPhPC and partially reproduced the channel's voltage dependence observed in patch-clamp experiments presented above. As previously 15 , mechanical stimulation was delivered by keeping one droplet stationary while periodically oscillating the second droplet in the axial direction with an amplitude of ±100 μm and frequency of 0.2 Hz. The most effective stimulation cycles had a shorter compression Scientific RepoRTS | (2018) 8:13607 | DOI:10.1038/s41598-018-31945-x phase and longer relaxation phase characterized with the duty cycle of 75% (the fraction of cycle time taken by the relaxation phase) 16 . Figure 2 shows typical traces that were continuously recorded at +100 mV ( Fig. 2a) and −100 mV (Fig. 2b) applied to the 'cis' compartment, i.e. droplet with V23T MscL-containing liposomes (see also Fig. 3d). The probability of seeing a channel opening event was higher at positive voltage (0.55 per cycle) than at negative voltage (0.28 per cycle). We should note that in DIBs V23T MscL opens to a variety of subconductive states, whose conductance on average was higher at +100 mV (Fig. 2b, inset). In terms of timing, opening events were well clustered near the pinnacle of compression phase, which lasts approximately 1 s (out of the entire 5 s cycle). An overlay of DIB current responses to seven selected cycles displaying openings is shown in Fig. 2c. Displacement in the positive direction ( Fig. 2d) signifies that the droplets are moved closer to each other, where they get distorted from the initial near-spherical shape and generate tension. As seen from the overlaid traces, V23T MscL activates  to various conductive levels and its dwell time in the open state (at +100 mV) stochastically varies between 100 and 500 ms. Although the period of time when super-threshold tension is acting on the channel is ~20% of the entire cycle duration (5 s), the cycles are identical and we presume that the open probability is proportional to the fraction of cycles in which openings were observed times the average channel conductance and open dwell time observed at that specific voltage.
Similar to patch-clamp data, increased voltage across DIB, negative or positive, increases the probability of seeing the opening event per cycle. V23T MscL, which is prone to opening to subconductive states 3 , also exhibits higher conductive states at higher voltages. These two factors, along with the dwell time in the open state, were taken into account in calculations of open probability (P o ). Since we never observed full openings of multiple channels in DIBs, we also assumed for simplicity that we deal with a single channel incorporated into the membrane. Increase of the presumed channel population to n will scale down P o but will not change the shape of voltage dependence. Figure 3a depicts P o as a function of voltage measured for DIBs and data from patch-clamp experiments extrapolated to the same tension. To compare two sets of data, we calculated the expected P o for V23T MscL in patches under applied voltage (−100 mV to 100 mV) using the intrinsic energy cost of opening (E o = 41 kT), the expansion area (ΔA = 18 nm 2 ) 3 and the capacitive (313.9 kT/V 2 ) and dipole (5.36 kT/V) voltage sensitivities of MscL derived from the above patch-clamp measurements. With these parameters, the tension that would produce the same P o in patches as observed in the DIBs system during the 0.5 s interval covering the maximum of compression phase (Fig. 2c) is estimated to be 7.34 mN/m. The estimated P o for MscL in patches and P o observed in DIBs (Fig. 3a) show good agreement over the whole range of applied voltages. The data were fitted with Eq. 2 and produced essentially coinciding lines despite the experimental scatter in the DIBs dataset. We have also re-calculated transition energies as functions of voltage from open probabilities obtained in DIBs and found good correspondence between the DIBs and patch-clamp data (Fig. 3b). Parabolic fits produced 288.4 kT/V 2 for the dielectric and 4.50 kT/V for the dipole contributions to voltage sensitivity.
The similarity between asymmetric voltage sensitivities of V23T MscL in patch-clamp and DIBs systems strongly suggests that channels added in in the form of liposomes into the cis compartment insert into the interfacial bilayer directionally as depicted in Fig. 3c,d. In the excised patch configuration, the channel points with its cytoplasmic side to the bath. Liposome incorporation into the DIB's cis compartment produces the same orientation as if the cis compartment represents the periplasmic side of the channel or the inner volume of the pipette. The general form of the energy E d of a dipole → p in a uniform electrostatic field → E is:

Interpretation of the dipolar and dielectric effects on
However, the external electrostatic field around the MscL channel is non-uniform due to the different geometries and dielectric properties of the pore, the protein, and the annular lipids subjected to distortion. That said, a more convenient approximation would be to consider the dipole as a system of two-point charges (a and b) at a distance d, where each of them is subjected to the external field according to the electrical potential in each of the locations (V a and V b ): We hypothesize that MscL's dipole arises from uneven charge distribution between the cytoplasmic and periplasmic domains, but the motion of these charges in the course of gating transitions (closed ↔ expanded ↔ open) is also accompanied by an electric field re-distribution due to protein flattening, pore expansion, hydration, and opening. Figure 4a visualizes the groups of charged residues and the distribution of electrostatic potential for E. coli MscL models in the closed and open states. The charged groups were taken in their default protonation states at neutral pH. The computation was done with the Particle Mesh Ewald (PME) algorithm in a vacuum, i.e., it reflects only the protein contribution to the electrostatics, unmitigated by the medium. The colour density indicates that the periplasmic side harbours the loops with the highest density of net negative charge, whereas the cytoplasmic side is richer in positive charges. These groups of charges clustered on opposite sides of the membrane form an effective dipole. We have to note, however, that all the smaller partial charges (e.g. hydroxyls), as well as the dipole moments of the transmembrane helices, also bring certain distributed contribution to the calculated net dipole moment. Approximating multiple partial charges and the resulting electrostatic field inside the protein with a single linear dipole is meant only to highlight the field density centres on the longitudinal cross-section of the system. This is a qualitative representation of the effective changes in charge and field distributions. The transition from the closed to the open state flattens the barrel essentially bringing the charges closer, but at the same time it brings the boundaries of the applied potential over the protein barrel closer together, thus increasing the intensity of the local transmembrane field. In addition, pore opening changes the distribution of electric field along the conductive pathway. The experiment (Fig. 1)  opening the domains that used to extend beyond the charged regions now pack deeper into the membrane, as the structural models of channel opening suggest 18,19 , and the charged clusters constituting MscL's dipole become exposed to a larger fraction of the external electric field that drops across the membrane segment harbouring the channel. In contrast, the taller and more 'protein-shielded' closed conformation (Fig. 4b, left) becomes more favourable at negative pipette potentials. We have constructed a cylindrical system consisting of the channel, lipid, and water parts, parametrized with "ballpark" geometric, dielectric and conductive properties approximating  (Table 1) show that the observed energetic effects of the dipole component can be reasonably estimated even in simplified cylindrical representation of the channel (Fig. 4b). In contrast to the dipole effect, the dielectric effect tends to maximize the exposure of high-dielectric components to electric field as it happens when part of a protein in the pore is displaced and substituted by water. For a cylindrical capacitor of radius r and thickness h filled with a medium of dielectric permeability ε, the dielectric energy E c can be expressed as a function of applied voltage: The total dielectric contribution to energy for a membrane-embedded MscL can be presented as three additive parts: E b related to flattening of the protein barrel, E a caused by distortion of annular lipids, and E i representing dielectric polarization of water that displaces the protein and fills the open pore interior.
The reason for the capacitive (dielectric) energy change for WT MscL is predominantly explained by the replacement of the low-dielectric pore-occluding part of the protein in the closed conformation with a volume of water in the open pore polarized by the part of the external voltage that drops across the transmembrane conductive pathway. Table 1 presents the key geometric parameters of the transition for WT and V23T MscL, dielectric properties of system components, and calculated energies associated with the transitions.

The tests for the local field effects introduced by membrane asymmetry. The above results
show that the energy of the MscL gating transition is affected by an externally applied electric field (Fig. 5). For V23T MscL the effect is especially strong because the geometric shape of this pre-hydrated channel in expanded/ subconductive states, which is highly populated in V23T, permits ion penetration into the vestibule which  . The septum is likely to be hydrated and it may take a minimal conformational transition that would generate a leakage, i.e., a subconductive state. The system subjected to voltage will likely favour subconductive states over the fully open state because in the latter case high pore conductance will 'shunt' the voltage across that narrow region thus reducing the local electric field. The voltage-dependence of V23T MscL is therefore likely imparted by a highly localized electric field. To test this notion and to answer the question of whether the channel feels not just the potential difference between the bulk solutions on the two sides of the membrane, but the superposition of both the external field and the intrinsic lipid-generated field, we studied voltage-dependent gating of V23T MscL in asymmetric DIBs. Such interfacial bilayers formed from lipid leaflets made of ester (DPhPC) and ether (DOPhPC) lipids bear a constant electrostatic bias created by the difference of interfacial dipole potentials of the two monolayers. Figure 6 illustrates the electrostatic effect of the permanent intrinsic electrical bias inside DIB revealed by mechanical oscillations periodically changing the area of the interface bilayer. Both traces in Fig. 6a,b are recorded under short-circuit conditions. When external voltage is 0, the displacement current predicted by the capacitive current formula = Δφ × I d C dt / c in is also zero in the case of a symmetric bilayer. When the bilayer is asymmetric and holds a permanent intrinsic bias, the current is detected. As was shown previously 14 , introduction of an external bias of ~130 mV compensates the intrinsic field in the asymmetric bilayer made of DPhPC and DOPhPC and therefore zeros the capacitive current. Figure 7 shows traces recorded in an asymmetric DPhPC/DOPhPC bilayer with directionally reconstituted V23T MscL. The channel is highly active at −100 mV and completely silent at +100 mV. This is a stark inversion of voltage dependence observed in symmetric DIBs (Fig. 3). The explanation comes from the consideration of electric potential profiles across asymmetric membranes given at the bottom of Fig. 7. The intrinsic bias of approximately 130 mV, which exists at zero applied voltage (Fig. 6b), is differentially influenced by the external voltage. The intramembrane voltage drop is amplified by negative (−100 mV) potential applied to the cis compartment and the channel gates with higher probability to higher conductive levels. The application of a positive 100 mV largely negates the already existing −130mV down to a −30 mV (the sum of both the externally applied voltage and the intrinsic bias), producing a value proven insufficient for the activation of V23T MscL in DIBs 15 . Therefore, the open probability of V23T MscL under given tension is augmented by the local potential dropping across the central part of the membrane between the boundary layers.

Discussion
MscL's transmembrane domain is composed of five pairs of interlocked TM1-TM2 helices (Fig. 4a). When subjected to membrane tension, the barrel expands in an iris-like manner through tilting of helical pairs [18][19][20] . The expansion of the barrel of WT MscL proceeds through a series of non-conductive conformations reaching the transition barrier positioned at about 70% on the in-plane expansion coordinate (ΔA) between the closed and fully open states 5 . The position and height of the rate-limiting barrier are apparently defined by the properties of the hydrophobic constriction lined by the L19 and V23 sidechains contributed from all five subunits. In the closed state, the constriction appears completely dehydrated (vapour-locked) and therefore leak-proof 4 . Increased tension produces more expanded states in which hydration of the hydrophobic constriction eventually becomes favourable 21 , thus creating a 'tipping point' on the energy profile leading to the open state 4 . The total transition energy includes terms related to the changes in protein-protein contacts, solvation, stretching of the periplasmic loops 22 , as well as transversal compression and bending of the annular lipids around the protein since the tilting of helices is accompanied by a substantial flattening of the barrel 23,24 . Solvation of the initially dehydrated hydrophobic pore stands out as a decisive factor since hydrophilic substitutions in that region dramatically decrease both the barrier and the energy difference between the closed and open states 3,4,25,26 . While the cooperativity of WT MscL gating is defined apparently by a single hydration event followed by essentially complete opening, the pre-hydrated pore of the V23T mutant allows for partial openings manifested as multiple subconductive states. The hydrophilic pore in the mutant is also pre-expanded in the resting state, and for this reason the apparent total lateral expansion is smaller than in the WT (18 nm 2 versus 20 nm 2 ). This initial volume of water stored deep in the pore may serve as the nucleation site for stronger hydration favoured by the applied transmembrane voltage. Indeed, water as a component with the highest dielectric constant in our system can be pulled into the region of strong electric field 27 located in the pore constriction near the middle of the membrane. Importantly, the voltage threshold at which the electrically-induced pores start accumulating in the lipid bilayer is in the range of 150-200 mV 28 , and the size of these pores is estimated to be comparable to MscL diameter (~ 3-5 nm) [29][30][31][32][33] . It appears that the effect of water treeing in cable insulators has precisely the same nature 34 .
The summary of gating energetics as a function of voltage for WT and V23T MscL is shown in Fig. 8A at two tensions. Since the voltage range accessible for patch-clamp or DIBs experiments is about ±120 mV, to illustrate expected energies in a fully energized state of the membrane 35,36 we extrapolated curves to ±200 mV. The top curve on panel a reflects the total opening energy (E o ) for the WT, which peaks at 58 kT at zero tension and zero voltage and asymmetrically bends down at ±200 mV. The result shows that WT is only slightly sensitive to voltage and will never open by physiological membrane potential at zero tension. To illustrate the scale of tension effect on the gating energy, we chose γ = 7.54 mN/m, corresponding to the peak tension in DIBs experiments (Fig. 2c,d). We should note that this tension is considerably below the WT activation threshold, however it does already shift the curve down to a new position with the energy maximum at 22 kT. This energy decrease (~36 kT) is due to the γΔA term, which reflects the WT MscL cooperative transition from the closed to the fully open state associated with the ΔA of 20 nm 2 . The electric contribution, responsible for bending the curve down at both ends, remains the same and extrapolations predict that at 7.54 mN/m membrane hyperpolarization to 200 mV would bring E o down to ~12 kT. This energy sets the open probability to ~10 −5 and thus may produce occasional openings.
For V23T MscL, the zero-tension E o has its maximum at 41 kT 3 , and the dielectric contribution produces a symmetrical parabolic energy dependence, which bends the curve down more substantially, reaching 29 kT at the ends. At γ = 7.54 mN/m, the entire curve slides down by γΔA = 33 kT, approximating the energy change due to opening to either a subconductive or fully open state with equal probability 3 . At this tension, the energy curve has its maximum at 8 kT, and the parabolas extrapolated to +200 mV predict that the transition energy may drop down to −5 kT, driving the channel conductive most of the time (99%) which, even at low-conductive substates, will become dangerous for the cell energetics. The zoomed-up view of electric field contributions for all energy curves with origins re-scaled to zero is presented on Fig. 8B. These effects that are negligible at low voltages typically used in electrophysiological recording (20-30 mV), become more pronounced at potentials typical for a fully energized state of the bacterial membrane (150-200 mV) 35 .
It is very feasible that the amino acid sequence of MscL can be further modified to design a channel that is even more sensitive to voltage than the V23T mutant. The green curves on Fig. 8 represent a hypothetical situation assuming that we can re-engineer the channel such that the barrel in the expanded state becomes 16% wider . This increases the volume of polarized water in the pore and further reduces the transition energy at +200 mV, which now becomes negative. This will drive spontaneous expansion and opening events even at no tension. Thinning of the partially permeable septum separating the upper and lower compartments by only 1 Å (20%) will further decrease the energy providing additional stabilization of the subconductive state.
The data presented in Fig. 7 show that the dielectric energy of water stored in the hydrated pore of V23T MscL is determined by the superposition of the external field and the drops of dipole potential across the interfacial layers. This provides a useful tool for the introduction of a constant field that may bias positions and sensitivities of membrane-embedded voltage sensors. In our case, this sensor is the water residing in the pore.
The data presented here depicts a consistent mechanistic picture that WT MscL gates between the open and closed states separated by an unusually large energy gap, ensuring that this osmotic safety valve stays shut to near lytic tension for the bacterial inner membrane. The overcoming of the large energy gap seems to involve a pseudo-phase transition at the nanoscale as a large pore is wetted. The effect of the large (physiological) bacterial membrane voltage appears to be minimized by two factors: (1) the 'dry' pore which essentially excludes water from penetrating and changing of dielectric properties inside the membrane, and (2) likely compensation of intrinsic protein electrostatics by the surrounding components.
Our first quantitative dataset shows that under normal voltages across the energized cytoplasmic membrane WT MscL resists spurious openings, and thus, is capable of preserving the barrier function of the inner membrane and bacterial energetics. The valine-to-threonine substitution in the constriction facilitates an opening transition and makes the channel significantly more prone to occupy subconductive states. This occurs at lower tensions when voltage approaches levels of a normally energized membrane. This substitution changes the character of voltage dependence, which is consistent with the primary role of water filling the pore and acting as a high-dielectric medium swelling the pore. This substitution makes the channel slightly toxic (as it dissipates transmembrane gradients) and for this reason polar substitutions for valine in this position are never found in any of the MscL orthologs. Threonine attracts water into the desolvated pore constriction and thus compromises the ability of the channel to hold high membrane potentials. However, the physical principle of MscL voltage modulation by a polar solvent can potentially be used for the engineering of molecular devices enabling us to adjust the tension sensitivity of sensors by voltage. Another notable aspect of this work is that here we provide the very first description of a surface dipole effect on a reconstituted channel imparted by the asymmetric lipid environment. This bears significance for both biology and bioinspired engineering because this result emphasizes the roles of interfacial dipole layers in setting the local electric field for the embedded molecule.

Methods
The patch-clamp experiments were performed as described previously 3,4,6 . The giant E. coli spheroplasts were generated from MJF465 E. coli cells 7 expressing either WT of V23T MscL. Patches were formed in symmetric 200 mM KCl, 90 mM MgCl 2 and 10 mM CaCl 2 in 10 mM Hepes-KOH buffer (10 mM, pH7.4). All recordings were done in excised inside-out patches under linear pressure ramps delivered from a modified HSPC-1 pressure clamp apparatus (ALA) using Axopatch 200B amplifier and PClamp 10 software.
The droplet interface bilayer (DIBs) recordings under periodic mechanical stimulation were performed exactly as in Ref. 14,15 . The aqueous droplets consist of a liposome suspension (2 mg/ml of either DPhPC or DOPhPC, Avanti Lipids), 500 mM of potassium chloride (KCl, Sigma Aldrich), and 10 mM of 3-(N-morpholino)propanesulfonic acid (MOPS, Sigma Aldrich) in deionized water (>18.2 MΩ.cm), pH 7. Hexadecane (99%, Sigma) is used as the oil phase. The hydrogel phase, used in the glass capillaries, consist of 40% (w/v) PEG-DMA contains 0.5% (w/v) Irgacure 2959, and is mixed with a 500 mM KCl and 10 mM MOPS, pH 7 electrolyte solution. The liposome and hydrogel solutions are prepared and stored as described elsewhere 37 . V23T MscL mutants, first generated and characterized by Anishkin et al. 3 , were isolated, reconstituted, and stored as described elsewhere 15,37 .
The DIB test setup is built and used as described by Najem et al. 15,37 . In the case of asymmetric studies, the droplet connected to the headstage contained DOPhPC, while the one connected to the ground contained DPhPC; salt concentration in both droplets were the same (500 mM KCl). We used Axopatch 200B and Digidata 1440 A (Molecular devices) in voltage-clamp mode for our electrical recording as described elsewhere 15,37 . The patch-clamp experiments were conducted as described elsewhere 3,6,18 .
The open probability of WT and V23T MscL in the course of the ramp stimulation in patch clamp was calculated as the fractional population current relative to the current at saturating pressure. In the case of DIBs experiments while we often deal with incomplete openings of single V23T channels to subconductive states, we still used the same approach of the fractional integral current to be compatible with patch-clamp estimates. For DIBs, the fractional population current was estimated as product of the average fraction of the time the channels were observed fully or partially open and the average relative single-channel conductance (as a fraction of a fully open single-channel current). It should be noted, that while the published experimentally estimated expansion area of V23T MscL from the closed to the fully open state (18 nm 2 ) is only slightly smaller than that of WT MscL (20 nm 2 ), the abundance of sub-conductive states with smaller expansion areas and lower energy costs 3 creates a spread of the conductive events towards the lower tension values. This creates shallower integral activation curve and an appearance of a smaller expansion area, resembling the effect of heterogeneity in the channel population 6 .

Data Availability
All data generated or analyzed during this study are included in the published article. Original data files are available upon request as needed.