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A Comparative Assessment of Effects of Major Mediators of Acute Phase Response (IL-1, TNF-α, IL-6) on Breathing Pattern and Survival Rate in Rats with Acute Progressive Hypoxia

A pressing issue of the day is the identification of therapeutic targets to suppress the “cytokine storm” in COVID-19 complicated by acute respiratory distress syndrome (ARDS) with concomitant hypoxemia. However, the key cytokine and its relative contribution to the pathogenesis of ARDS, which leads to high mortality, are unknown. A comparative assessment of the effect of elevated systemic levels of pro-inflammatory cytokines IL-1β, TNF-1α and IL-6 on the respiratory patterns and survival rate in rats was carried out under progressively increasing acute hypoxia. Increasing hypoxia was simulated by a rebreathing method (from normoxia to apnea). The recorded parameters were the breathing pattern components (tidal volume and respiratory rate), minute ventilation (MV), oxygen saturation, apnea onset time, and posthypoxic survival rate. A comparative analysis was carried out under mild, moderate and severe hypoxia (at FIO2 = 15, 12 and 8%, respectively). It was shown that increasing hypoxia was accompanied by an acute suppression of the compensatory elevation of MV in rats with increased systemic levels of IL-1β and TNF-1α. By contrast, IL-6 caused an intensive elevation of MV with increasing hypoxia. Acute hypoxia (FIO2 < 8%), in all experimental series, was accompanied by an impairment of the respiratory rhythm up to the development of apnea. Posthypoxic breathing restoration (survival rate) was 50% with IL-1β and TNF-1α and only 10% with IL-6. The obtained results indicate that the elevated IL-6 level, despite the absence of respiratory disorders at the initial stage of the developing pathologic process, leads to a higher mortality in rats compared to IL-1β and TNF-1α. This allows considering IL-6 as an early prognostic biomarker of a high risk of mortality under severe hypoxemia.

The universal nonspecific response of the organ ism to the impact of damaging exo and endoge nous factors caused by the invasion of a viral or bacterial infection is a systemic inflammatory response [1]. The development of a systemic inflammatory response relies on the uncontrolled massive production of acute phase response medi ators, including such pro inflammatory cytokines as interleukin 1β (IL 1β), interleukin 6 (IL 6), tumor necrosis factor alpha (TNF α), and many other biologically active substances [2]. In turn, the production of a large amount of pro inflammatory mediators leads to the activation of immune cells, which release new quanta of mediators due to the presence of an uncontrolled positive feedback between these processes [3]. A vicious circle thus arises, causing tissue destruction in the focus of inflammation, the spread of the response to neigh boring tissues, and the release of pro inflammatory cytokines into the bloodstream. As a result, inflam mation becomes systemic, generalized, covering the whole organism.
Currently, it has been proven that the massive cytokine releasing cascade, called "cytokine storm", is associated with multiple organ failure [4,5], with the lungs being, in most cases, a target organ [6]. Hyperactivation of the immune response can cause the development of acute respiratory dis tress syndrome (ARDS) characterized by pneumo nia, exudative lesions, decreased elastic properties of the lungs, pulmonary embolism, inadequate relationship between ventilation and perfusion of pulmonary alveoli, and impaired gas exchange, which ultimately threatens respiratory arrest and lethal outcome [7 ]. There is evidence that the severity of the clinical course of the disease and in hospital mortality of patients are aggravated by the main manifestation of ARDS, a rapidly increasing hypoxemia [8,9], which causes the additional expression of circulatory pro inflammatory cyto kines, such as TNF α, IL 1β and IL 6 [10]. Clini cal observations have found a close link between the degree and duration of hypoxemia and mortal ity in patients with ARDS [11].
There is also evidence that, among the numer ous cytokines released during the systemic inflam matory response, one of the main mediators of viral pneumonia is IL 1β, which is used as a bio marker to classify the severity of the disease [7]. IL 1β is the most studied pleiotropic cytokine whose effects are similar to those of TNF α, also referring to the main pro inflammatory cytokines, which in turn promotes the production of other cytokines, including IL 6 [12]. This cytokine, having both pro inflammatory and anti inflam matory properties, is expressed by stromal and immune cells, while its release is regulated, spe cifically, by IL 1β and TNF α [13]. In addition, any inflammatory stimulus increases the expres sion of IL 6, which can also serve as a trigger for IL 1β and TNF α. Large scale clinical studies conducted on hospitalized patients diagnosed with COVID 19 found that, in contrast to the mild form, the severe and critical course of the disease is characterized by a high IL 6 expression, which correlates with respiratory failure, second ary infections, and mortality. Elevated IL 6 levels were also associated with the risk of death in patients with ARDS without COVID 19 [6,14].
Currently, in the context of the coronavirus dis ease (COVID 19) pandemic, novel approaches to the therapy of systemic inflammation are being intensively developed, such as inhibition of the production and activity of pro inflammatory cytokines, stimulation of the synthesis of anti inflammatory cytokines, and searching for thera peutic targets to suppress the cytokine storm. In addition, given the high rate of in hospital mor tality, there is a need to identify early predictive biomarkers to stratify hospitalized patients in accordance with the probability of mortality risk.
An important criterion for the diagnosis, deci phering the pathogenesis, determining the prog nosis and therapeutic tactics of treatment is the set of individual components of the respiratory cycle-depth, respiratory rate, minute lung venti lation, etc. (breathing pattern), which changes with the onset and progression of respiratory dis eases. However, the relative contribution of IL 1β, IL 6, and TNF α to changes in the breathing pat tern against the background of progressive hypox emia has hardly been studied.
In connection with the aforesaid, this work aimed to compare the effect of the key pro inflammatory cytokines IL 1β, IL 6, and TNF α on the breathing pattern, blood oxygenation, and the rate of mortality during increasing hypoxia.

MATERIALS AND METHODS
The work was carried out on animals from the Pavlov Institute of Physiology's Collection of Laboratory Mammals of Varied Taxonomic Affil iation supported by the Russia's Bioresource Col lections program. All experimental procedures that involved animals complied with the ethical standards approved by legal acts of the A study was carried out in acute experiments on 58 Wistar rats weighing 280-300 g, and anesthe tized with urethane (1000 mg/kg, i.p.). The depth of anesthesia was monitored by the degree of the corneal reflex manifestation. Rats were divided into 5 groups: group 1-intact animals (vivarium control), group 2-control animals administered with 1 mL of saline (0.9% NaCl), group 3-ani mals administered with interleukin 1β (recombi nant human IL 1β, State Institute of Highly Pure Biopreparations, Russia; 500 ng/mL of saline), group 4-animals administered with tumor necro sis factor α (TNF α, Sigma, USA; 40 mg/kg), group 5 -animals administered with interleukin 6 (IL 6, Sigma, USA; 50 μg/kg).
After recording the baseline (normoxic) values, the agents under study were injected into the fem oral vein, and after 70 min, the rats were switched to breathing with a hypoxic mixture with a pro gressive decline of the oxygen content (from nor moxia to a complete respiratory arrest) using the rebreathing method [15]. The onset time and duration of hypoxic apnea, as well as the fraction of inspired oxygen in the gas mixture (F I O 2 ), and posthypoxic survival rate (lethality) (%) vs. the control group were recorded. The respiratory arrest for 1 min was equated to death of the ani mals. During the experiment, the studied parame ters were recorded continuously at every minute, and a comparative assessment was carried out at F I O 2 = 15, 12 and 8%.
The main parameters of external respiration, such as the volume velocity of the inspiratory flow (Vi), tidal volume (V T ), and respiratory rate (RR), were recorded by the pneumotachographic method. The minute ventilation (MV) was calcu lated as a product of V T and RR. To measure esophageal (a surrogate of intrathoracic or pleu ral) pressure (P es ), the balloonographic method was used. Blood oxygenation (SpO 2 , %) was determined using the UT100 veterinary pulse oximeter (Zoomed, Russia). F I O 2 in the inspired gas mixture was measured by the PGK 06 oxygen analyzer (Insovt, Russia), and the fraction of car bon dioxide in the inspired gas mixture (F iCO2 ) was measured with the MAG 6P multicompo nent gas analyzer (Exis, Moscow). After the experiment, animals were euthanized by an over dose of urethane.
The pneumotachogram and P es pressure signals were digitized and stored on a PC HDD using a hardware software complex created on the basis of the Biograf 7 biological data collection device (St. Petersburg State University of Aerospace Instrumentation, Russia).
Statistical data analysis was carried out using Statistica 10.0 (Windows) and Microsoft Office Excel 2020 software packages. To check the sam ple for normal distribution, the Kolmogorov-Smirnov test was used; the resulting significance level (p ≥ 0.2) indicated that the grouped data samples followed a normal distribution. Then, the values before and after drug the injection of the above agents were assessed using the paired Stu dent's t test and two way (ANOVA) for the hypoxia-normoxia and control-agent factors. Differences were considered significant at p < 0.05; the data in the figures and table are pre sented as M ± SE.

RESULTS
As follows from Table 1, under normoxic con ditions, significant changes in all the studied parameters were only observed with an increase in IL 1β, while TNF α caused an increase in V T and MV, and IL 6 did not affect the parameters of external respiration at all.
A similar dynamics of MV under hypoxia was also observed in rats after IL 1β and TNF α administration, with the differences relating to the degree of increment, which was significantly lower than in control rats. For instance, in rats adminis tered with IL 1β, MV at the level of 15% O 2 increased by 20 ± 7%, at 12% О 2 by 18 ± 8%, and at 8% О 2 , there was no increment in MV (its value corresponded to those under normoxia) (Fig. 1c). In the group administered with TNF α, at 15% O 2 , MV increased by 10 ± 3%, at 12% O 2 by 18 ± 4%, respectively (p > 0.05); at 8% O 2 , there was no increase in MV. A decrease in MV incre ments under hypoxia in rats with elevated IL 1β and TNF α levels resulted from decreased incre ments in inspiratory P es and V T fluctuations (Figs. 1a, 1d). RR under mild and moderate hypoxia changed insignificantly. In both control and experimental groups, a significant decrease in RR was only observed under severe hypoxia (8% O 2 ) (Fig. 1b).
In rats with an increased systemic IL 6 level, the opposite dynamics of the respiratory parame ters was observed. As hypoxia increased, there was a significant increase in the MV increment rela tive to normoxia. For instance, at 15% O 2 , the MV increment was 20 ± 3% (p > 0.05), at 12% O 2  . 1. Increments in external respiration parameters to increasing hypoxia in rats with elevated IL 1β, TNF α and IL 6 lev els. Data are presented as the M ± SEM (n = 12 in each group); * р < 0.05 vs. normoxia, # р < 0.05 vs. control (NaCl). X axisgroups studied, Y axis-increments in % of normoxia: (a)-tidal volume, (b)-respiratory rate, (c)-minute ventilation, (d)esophageal pressure. A sharp weakening of the ventilatory response to increasing hypoxia was observed with an increase in sys temic IL 1β and TNF α levels. IL 6 did not inhibit the ventilatory response to increasing hypoxia, causing only an insignifi cant decrease in the respiratory rate and not affecting other parameters compared to the control animals. 50 ± 9%, and at 8% O 2 72 ± 13%, respectively (p < 0.05). At the same time, a significant increase in V T and P es increments was observed, exceeding the corresponding values in the control group under severe hypoxia (Figs. 1a, 1d). RR decreased to a lesser extent than in animals of other groups. Blood oxygenation (SpO 2 ) with increasing hypoxia decreased in all the studied groups com pared to the normoxic control (95%). The most pronounced decrease in SpO 2 (up to 46%) was observed in rats administered with IL 1β under acute hypoxia, while the dynamics of SpO 2 falling with increasing hypoxia was comparable to the control. In rats administered with TNF α, SpO 2 stabilized at the 75% level, regardless of the degree of an increase in hypoxia (Fig. 2). IL 6 also caused a decrease in SpO 2 with increasing hypoxia: under the maximum degree of hypoxia (F I O 2 = 8%), SpO 2 was 66%.
Increasing acute hypoxia (F I O 2 < 8%), in all the experimental series, was accompanied by an impairment of rhythmic breathing, turning even tually into apnea. However, it should be noted that respiratory arrest in the studied groups occurred under different degrees of hypoxia: in the control group, apnea was observed at F I O 2 3-4% О 2 , in the groups administered with IL 1β at 7-8% О2, with TNF α at 3-4% O 2 , with IL 6 at 6-7% O 2 . The posthypoxic restoration of the inspiratory activity (survival rate) in the groups with elevated IL 1β and TNF α levels decreased 2 fold compared to the control, i.e. respiration was restored only in 50% of the experimental ani mals, while in the control group the survival rate was 100%. A highest mortality rate was found in rats administered with IL 6, in which a sponta neous restoration of breathing in the posthypoxic period was observed only in 10% of cases.

DISCUSSION
Comparative analysis of respiratory effects of the three key proinflammatory cytokines showed that in rats administered with IL 1β and TNF α, a compensatory increase in MV decreases in response to hypoxic exposure compared to the control, while IL 6 has an opposite effect, i.e. it does not depress, but, instead, increases the incre ment in MV as the hypoxic exposure rises. Partic ularly sharp differences are observed under the effect of a severe degree of hypoxia (8% O 2 ).
A negative dynamics of MV increments within the individual groups (control, IL 1β, TNF α) was observed against the background of a positive dynamics of P es and V T , being caused by a signifi cant decrease in RR. The most dramatic decrease in RR was observed under severe hypoxia. At the same time, with a positive dynamics of P es and V T in the groups with elevated IL 1β and TNF α sys temic levels, an increase in these parameters was significantly lower than in the control group. Under severe hypoxia, against the background of IL 6 action, RR decreased to a lesser extent than in the control group, and the V T increment corre sponded to the control level. As a result, under a combined effect of severe hypoxia and IL 6, MV was higher than in the control and other experi mental groups.
To date, it has been established that the cyto kines IL 1β and TNF α are involved in neuroim mune interactions in the brain regions responsible for the central regulation of breathing. This is fur ther supported by the data indicating that IL 1β and TNF α receptors are found in the solitary tract nucleus and ventrolateral part of the medulla oblongata [12,16,17]. It was also found that IL 1β and TNF α are expressed on sensory cells of the carotid body, reducing the sensitivity of glomus cells to oxygen deficiency [18]. Therefore, IL 1β and TNF α mediated neuroimmune interac tions can also be involved in the peripheral mech anisms of respiratory control that modulate the response to hypoxia and arterial blood saturation with oxygen. As shown in the acute pneumonia model, levels of IL 1β and TNF α, but not IL 6, increase in the bronchoalveolar fluid and lung tis sue [19].
In addition, in contrast to IL 1β and TNF α, IL 6 has not only pro inflammatory, but also anti inflammatory properties [20]. Moreover, IL 6 is classified as a myokine expressed by muscle fibers during exercise [21]. IL 6 was shown to exhibit anti inflammatory properties during muscle con traction, while an increased IL 6 plasma level additionally causes the expression of more power ful anti inflammatory cytokines, such as IL 10 and an IL 1β receptor antagonist, and at the same time inhibits the synthesis of the pro inflamma tory cytokine TNF α [21]. These data can to some extent account for the differences we obtained in the responses of external respiration to increasing hypoxia between the groups with increased IL 1β and TNF α systemic levels and the group with an elevated IL 6 level.
Our present results indicate that IL 6 affects the respiratory system to a lesser extent than IL 1β and TNF α. It was found that it does not have a direct negative effect on the main inspiratory muscle, diaphragm [22]. Our results are consis tent with these data, since under normoxic condi tions IL 6 did not cause a decrease in P es which reflects the total respiratory muscle effort. Increasing hypoxia, against the background of IL 6 administration, caused enhanced respiratory muscle contractions, an increase in P es , and a corresponding increase in MV. However, despite the absence of respiratory failure and a significant decrease in SpO 2 in this group of rats, the survival rate was only 10% compared to the control, i.e. 5 times lower than in other experimental groups. The facts we discovered can be explained as fol lows. It is well known that, to detect hypoxemia in clinical practice, a noninvasive method of pulse oximetry (SpO 2 ) is often used, which, however, reflects not a true oxygen tension in arterial blood (PaO 2 ), but a hemoglobin saturation with oxygen. According to the physiological principles [23], oxygen release from hemoglobin into the blood depends primarily on the position of the S shaped oxyhemoglobin dissociation curve. Hypocapnia and respiratory alkalosis that result from hyper ventilation shift the dissociation curve leftward and increase the affinity of hemoglobin for oxy gen, thus hampering oxygen release into the blood, and this can explain a high SpO 2 level at a very low PaO 2 [24]. A similar discrepancy between the hypoxemia severity and minor respi ratory discomfort, the so called silent or happy hypoxia, was found in patients with COVID 19 [8], as well as in healthy people staying under high altitude conditions, when hypocapnia sig nificantly shifts the oxyhemoglobin dissociation curve and increases hemoglobin saturation with oxygen [25]. Based on these facts, it can be assumed that one of the probable causes of high mortality in rats administered with IL 6 may be a severe arterial hypoxemia, despite the retention of SpO 2 and the absence of signs of respiratory fail ure [26]. In rats with elevated IL 1β and TNF α levels, a significant depression of lung ventilation was observed under increasing hypoxia; in these conditions, hypoventilation is inevitably accom panied by hypercapnia [27]. It is possible that the lack of hypocapnia in these groups contributed to a higher survival rate of the animals.
The revealed differences in the SpO 2 level under increasing hypoxia between the studied groups suggest the presence of multiple mecha nisms for the tested cytokines to affect the devel opment of hypoxemia. There is evidence that, in addition to changing the position of the oxyhemo globin dissociation curve, cytokines may directly affect the heme group of hemoglobin [28], intrapulmonary shunting [29], ventilation perfu sion ratios and diffusion capacity of the lungs [30], intravascular blood microclotting [31], etc.
In addition, the higher mortality rate of animals administered with IL 6, despite the lack of respi ratory decompensation, may have been associated with its negative impact not on the respiratory sys tem, but on the cardiovascular system. It has been shown that microinjection of IL 6 directly into the solitary tract nucleus modulates input (affer ent) signals from baroreceptors, being thus involved in the regulation of the cardiovascular system [32].
Thus, IL 1β, TNF α and IL 6, under condi tions of progressively increasing hypoxia, have different modulatory effects on the breathing pat tern, hemoglobin oxygen saturation, and the sur vival rate of rats after hypoxic apnea. IL 1β and TNF α cause acute respiratory failure, which develops already at an early developmental stage of the pathological process due to suppression of a compensatory increase in lung ventilation to an increasing hypoxic stimulus, mediated by a impairment of the central and peripheral regula tory mechanisms of respiration. IL 6, despite the lack of obvious signs of respiratory decompensa tion, in contrast to IL 1β and TNF α, leads to a greater mortality in rats, probably due to a more rapid increase in arterial hypoxemia. These find ings can serve as a basis for assessing the systemic IL 6 level as an early prognostic biomarker of the likelihood (high risk) of death under severe forms of acute respiratory distress syndrome.

FUNDING
This work was supported by the Russian federal budget allotted to Pavlov Institute of Physiology (Russian Academy of Sciences) for the imple mentation of the governmental assignment.