Fever-range whole body hyperthermia leads to changes in immune-related genes and miRNA machinery in Wistar rats

Abstract

Objective

Fever is defined as a rise in body temperature upon disease. Fever-range hyperthermia (FRH) is a simplified model of fever and a well-established medical procedure. Despite its beneficial effects, the molecular changes induced by FRH remain poorly characterized. The aim of this study was to investigate the influence of FRH on regulatory molecules such as cytokines and miRNAs involved in inflammatory processes.

Methods

We developed a novel, fast rat model of infrared-induced FRH. The body temperature of animals was monitored using biotelemetry. FRH was induced by the infrared lamp and heating pad. White blood cell counts were monitored using Auto Hematology Analyzer. In peripheral blood mononuclear cells, spleen and liver expression of immune-related genes (IL-10, MIF and G-CSF, IFN-γ) and miRNA machinery (DICER1, TARBP2) was analyzed with RT-qPCR. Furthermore, RT-qPCR was used to explore miRNA-155 levels in the plasma of rats.

Results

We observed a decrease in the total number of leukocytes due to lower number of lymphocytes, and an increase in the number of granulocytes. Furthermore, we observed elevated expressions of DICER1, TARBP2 and granulocyte colony-stimulating factor (G-CSF) in the spleen, liver and PBMCs immediately following FRH. FRH treatment also had anti-inflammatory effects, evidenced by the downregulation of pro-inflammatory macrophage migration inhibitor factor (MIF) and miR-155, and the increased expression of anti-inflammatory IL-10.

Conclusion

FRH affects the expression of molecules involved in inflammatory processes leading to alleviated inflammation. We suppose these effects may be miRNAs-dependent and FRH can be involved in therapies where anti-inflammatory action is needed.

Keywords:

• Fever-range whole body hyperthermia
• miRNA machinery
• miRNA-155
• cytokines
• immunomodulation

1. Introduction

Fever is defined as a rise in body temperature (increase in body heat) upon disease. As part of the acute phase response, fever is almost invariably accompanied by uncomfortable sickness symptoms and behaviors, notably lethargy, depression and aches [1]. Physiologists ascribe fever to an upward resetting of the thermoregulatory set point induced by either infectious agents or factors synthesized by the host. The purpose of the increase in body temperature that is associated with fever remains the most poorly understood aspect of the acute inflammatory response.

To investigate the significance of the thermal component itself, fever-range hyperthermia (FRH) has been employed. There are many in vitro research that presents the effect of high ambient temperature on various cell lines [2–4]. In our previous in vitro study, we described the effect of hyperthermia on macrophages. We have found that hyperthermia may affect therapy in which mistletoe extract is involved. We showed that the treatment of macrophages (RAW 264.7 cells) with elevated temperatures (39 °C and 41 °C) stimulated the expression of pro-inflammatory cytokines, including interleukin (IL)-6 and IL-1β [5]. Thus, we indicated its immunomodulatory effects. However, that in vitro model did not reflect the response of a whole body to FRH. Therefore, in this study, we employed rats to investigate molecular mechanisms triggered by the heat.

Among organs that are affected by FRH, the liver deserves special attention. This is an organ that displays temperature-dependent metabolic rate changes [6], and releases various regulatory molecules, including cytokines and complement proteins [7]. Similarly, the spleen plays an important role in the modulation of the immune system. This is a place for the differentiation and activation of T and B cells [8].

The count of white blood cells and the distribution of these cells in a body is regulated by many factors including G-CSF that regulates granulocytopoiesis and macrophage migration inhibitory factor (MIF). MIF is a cytokine released by a number of cell types including, monocytes, macrophages and T cells. Research has shown its important role as a regulator of innate immunity and its pro-inflammatory properties [9]. The expression of MIF may be induced by various pro-inflammatory factors including cytokines, microbial products and in response to stress [4,9].

Another important pro-inflammatory factor is interferon-γ (IFN-γ) which acts as the first line of defense against viral infection in mammals. IFN-γ can act directly on infected cells or is able to induce indirect anti-viral response through modulating the differentiation and maturation of T cells and B cells [10]. IFN is well-known as a mediator of adaptive immunity but it may also stimulate innate immunity through the activation of macrophages [11].

To counteract the damage caused by inflammatory processes, anti-inflammatory agents such as IL-10 are released by organisms. Activation of IL-10 receptors leads to inhibition of pro-inflammatory mediators, decrease of phagocytosis and the release of anti-inflammatory molecules such as interleukin-1 receptor antagonist, soluble tumor necrosis factor α (TNFα) receptor and IL-27 [3,12].

Although, the role of G-CSF, MIF, IFN-γ and IL-10 in inflammatory processes is established, it is not known whether FRH can affect these factors. Furthermore, it is still not known how the network of these cytokines is regulated. Recently, the role of miRNAs is postulated [13–16]. Increasing evidence suggests that the liver can be a source of micro RNAs (miRNAs) [7]. Although miRNAs are intracellular regulators of gene expression, they can be secreted into the extracellular matrix and body fluids, and delivered to distant cells to trigger systemic effects [17].

miRNAs are small (∼22 nt), single-stranded, non-coding RNAs that regulate gene expression through base-specific pairing between the seed region of the mature miRNA and the 3′-untranslated regions (3′-UTR) of target mRNAs [18]. The biogenesis of miRNAs begins in the nucleus, in which long RNA transcripts termed primary miRNAs (pri-miRNAs) are excised. pri-miRNAs are then processed and final miRNAs are released. Dicer1 and Tarbp2 are key to this process [7], and regulate the expression and functioning of all miRNA molecules including miRNA-155 [19]. miRNA-155 has been extensively investigated due to its role in immune defense. It may be expressed by a wide spectrum of cells and it regulates both innate and adaptive immunity, including monocytes, macrophages, dendritic cells, B cells and T cells [20].

In this study, we developed a fast rat model of FRH induced by infrared radiation. Our study aimed to investigate the effect of FRH on the expression of cytokines that regulate inflammation. We further analyzed the effects of FRH on the expression of miRNA machinery molecules (DICER1 and TARBP2) and miR-155.

2. Materials and methods

2.1. Experimental animals

Male Wistar Cmd:(WI)WU rats aged 4–5 weeks and weighing 120–150 g were purchased from the Mossakowski Medical Research Centre of the Polish Academy of Sciences (Warsaw, Poland). Rats were acclimatized for 14 days and housed at constant relative humidity (60 ± 5%) and temperature (22 ± 1 °C), with a 12:12 h light–dark cycle (light at 7 am). Food and water were provided ad libitum. All procedures were approved by the local Bioethical Committee for Animal Care (permission no. 49/2020).

2.2. Temperature measurements

To monitor deep body temperature (Tb), animals were implanted intraabdominally with temperature-sensitive miniature biotelemeters (PhysioTels model TA10TA-F40; Data Sciences International, St. Paul, MN) under sterile conditions according to manufacturer’s recommendations. Prior to the implantation, rats were anesthetized with a mixture of ketamine (Biowet, Puławy, Poland)/xylazine (ScanVet, Gniezno, Poland) (87 mg/kg and 13 mg/kg, respectively) injected intramuscularly. Following shaving and sterilization of a small surgical area, an incision was made in the skin and muscles of the abdomen. A miniature temperature-sensitive telemetry device was then placed into the peritoneal cavity and the abdomen and skin were independently sutured. All surgical procedures were performed a minimum of 10 days before experimentation.

2.3. Treatment of rats with fever-range whole body hyperthermia

FRH was induced using an infrared lamp and heating pad (Data Science International). Prior to induction, rats were anesthetized with a mixture of ketamine/xylazine injected intramuscularly. Animals were placed on their backs on a heating pad and temperature probes were introduced per rectum to monitor deep body temperatures during FRH. Following assessment of the initial body temperature, the infrared Sollux lamp Breuer (150 W 230 V, Sollux, Berlin, Germany) was switched on and heating was performed for 5 min with a lamp placed 30 cm from the body surface. Upon exceeding a body temperature equal to 38.1 °C, the lamp was switched off. To maintain a constant temperature of 39 °C for 1 h, a heating pad was employed. If the deep body temperature decreased to 38.7 °C, the lamp was switched on for additional 30s. Following heating, during restoration to the physiological range, the deep body temperature was measured using a biotelemetry system (Data Science International).

2.4. Tissue sample collection from rats

Tissue samples were collected immediately after FRH treatment (FRH-t₀) or 24 h post-FRH (FRH-t₂₄). The Liver and the spleen samples (whole glands) were dissected from anesthetized rats, rinsed twice with cold sterile PBS, dried and then frozen in liquid nitrogen. Samples were stored at −80 °C prior to analysis.

2.5. Blood sample collection and plasma isolation

Blood was collected into EDTA-treated tubes following cardiac puncture. Plasma separations were performed through centrifugation of whole blood samples for 10 min at 1000 ×g. Samples were transferred into fresh tubes and stored at −80 °C prior to assessment.

2.6. Evaluation of blood cell counts in rats

The total number of blood leukocytes, lymphocytes and granulocytes were investigated using an Auto Hematology Analyzer BC-2800Vet (Mindray, Shenzhen, China) according to the manufacturer’s recommendations.

2.7. Peripheral blood mononuclear cells isolation

Density gradient centrifugation was used for the isolation of peripheral blood mononuclear cells (PBMCs). Following the collection of whole blood, samples were diluted 1:1 with sterile PBS at room temperature. Dilutions were layered onto Lymphocyte Separation Medium (BioWest, Nuaillé, France) in 15 ml tubes, centrifuged (35 min, 400 × g, room temperature) and the PBMCs were collected. To remove the separation medium, cells were washed with sterile PBS and centrifuged for 5 min at 400 × g. Cells were resuspended in Fetal Bovine Serum (Merck KGaA, Darmstadt, Germany) supplemented with 10% dimethyl sulfoxide (DMSO), frozen and stored in liquid nitrogen prior to their use.

2.8. Total RNA extraction

For RNA extraction, liver and spleen tissues (100 mg) or PBMCs were lysed in PureZOL™ RNA Isolation Reagent (Bio-Rad, Hercules, CA) with mechanical disruption for tissue samples. mRNA extraction was performed according to the Chomczynsky and Sacchi method [21]. The final concentration of total mRNA in the samples was measured using a Take3 Micro-Volume Plate on a Synergy HT Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT).

2.9. RT-qPCR

cDNA synthesis was performed on 1000 ng of total RNA using iScript™ Reverse Transcription Supermix for RT-qPCR. qRT-PCRs were performed using iTaq Universal SYBR® Green Supermix and PrimePCR™ SYBR® Green Assays with a final volume of 10 µL. Unique assay IDs of the primers are shown in Table 1. Test samples were run in triplicate using the CFX Connect Real-Time PCR Detection System. Melt curve analysis was performed as a control for specificity. Standard curves were prepared for both target and reference (Actb) genes. Calibrator-normalized quantifications were performed using CFX Manager Software 3.1. Reactions were repeated at least twice. All reagents were purchased from Bio-Rad.

Table 1. Unique Assay ID of primers used for RT-qPCR.

Gene name	Gene/PROTEIN symbol	Unique Assay ID	Cat. No
Actin	Actb/ACTB	qRnoCID0056984	Cat. No. 10025636
RISC-loading complex subunit TARBP2	Tarbp2/TARBP2	qRnoCED0004684	Cat. No. 10025636
Dicer1	Dicer1/ DICER1	qRnoCID0006103	Cat. No. 10025636
Macrophage migration inhibitory factor	Mif/ MIF	qRnoCED0007731	Cat. No. 10025636
Granulocyte colony-stimulating factor precursor	Csf3/ G-CSF	qRnoCED0001885	Cat. No. 10025636
Interferon gamma	Ifng/IFN-γ	qRnoCID0006848	Cat. No. 10025636
Interleukin-10	IL10/ IL-10	qRnoCID0005930	Cat. No. 10025636

2.10. miRNA extraction and expression

Circulating miRNA molecules were isolated from 200 µL of plasma using the miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. TaqMan®microRNA RT Kits were used to reverse transcribe cDNA of TaqMan®MicroRNA assays for mmu-miR-155 and U6 snRNA as an internal reference (Table 2) [22]. qPCRs were performed in a final volume of 20 µL and included 1.33 µL of RT product, 1X TaqMan™ Universal PCR Master Mix II, no UNG and probe mix (Thermo Fisher Scientific, Waltham, MA). Samples were analyzed in triplicate using the CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, CA). Relative expression levels were obtained through normalization to pre-FRH samples. Values were calculated using CFX Manager Software 3.1 (Bio-Rad, Hercules, CA).

Table 2. Unique primer IDs for miRNA analysis by RT-qPCR.

	ID No.	Cat. No
RT-U6 snRNA	001973	PN4427975
RT-mmu-miR-155-5p	002571	PN4427975
TM-U6 snRNA	001973	PN4427975
TM-mmu-miR-155-5p	002571	PN4427975

2.11. Statistical analysis

Relative miRNA expression level and gene expression levels were calculated and compared using the 2^−ΔΔCT method. All the data analyses were performed three times with three technical repetitions for each one. Multiple group comparisons were performed using a one-way analysis of variance (ANOVA) followed by the Tukey test. For calculations, analysis, and results visualization GraphPad Prism version 8 (GraphPad Software Inc., La Jolla, CA) statistical software was used. The statistical standard of significance was set at p < .05.

3. Results

3.1. Establishment of a rat model of fever-range whole body hyperthermia using infrared radiation

To investigate the effects of heat in rats, we developed a whole body FRH model. The initial average deep body temperature of the animals was 36.77 ± 0.57 °C (mean ± SD). The average time of heating animals with an infrared lamp to the level of 38.10 °C was 5 ± 2.55 min. Treatment of rats with an infrared lamp increased the body temperature to 39.00 °C within next 3 ± 2.35 min since switching off the infrared lamp, which was maintained for 1 h. The body temperature did not fall below 38.94 ± 0.05 °C and did not exceed 40.08 ± 0.30 °C (Figure 1). Upon cessation of heating, the average time required for normalization to physiological levels was 35 min.

Figure 1. Changes in body temperature within 100 min of FRH. The shadowed background denotes the mean body temperature during 60 min of FRH. The additional 40 min represents the time required for the restoration of the body temperature to physiological levels. FRH: Fever Range Hyperthermia treatment; NT: control group, n: sample size per group.

3.2. Fever-range hyperthermia decreases the number of white blood cells

We next evaluated the effects of FRH on WBC counts. We observed a significant decrease in total WBC numbers post-FRH in rats (p < .05; Figure 2(A)). After 24 h, the number of WBCs still decreased, though these changes were not statistically significant (p = .44).

Figure 2. Effects of FRH on white blood cell (WBC) counts. (A) White blood cell count, (B) total number of lymphocytes; (C) lymphocytes as a percentage of WBCs; (D) total number of granulocytes; (E) granulocytes as a percentage of WBCs; (F) total number of monocytes and (G) percentage of monocytes in the peripheral blood of FRH-treated rats. Data are the mean ± SEM Asterisks indicate significant differences (**p < .01, *p < .05). NT: control animals (n = 6), FRH-t₀: samples collected directly after FRH treatment (n = 6), FRH-t₂₄: samples collected 24 h post-FRH treatment (n = 5). n indicates the sample size per group.

Lymphocyte numbers were found to decrease in response to FRH (Figure 2(B)), which occurred directly following FRH treatment (p < .05) and continued for 24 h (p < .01). The percentage of lymphocytes in WBCs decreased after 24 h (p < .01), compared to the control group (Figure 2(C)). Our analyses revealed that restoration of total WBC numbers after 24 h was related to a rise in the number of granulocytes in comparison to the control group and rats directly post-FRH (p < .01 and p < .05, respectively; Figure 2(D)). Similarly, we observed substantial changes in the percentage of granulocytes only after 24 h (p < .01; Figure 2(E)).

Although the number of monocytes decreased following FRH, the decline was not statistically significant (p = .47; Figure 2(F)). Similarly, differences in the percentage of monocytes in response to FRH were observed (Figure 2(G)). Collectively, these data suggest that FRH leads to substantial growth in granulocyte numbers while the number of lymphocytes decreases.

3.3. Fever-range hyperthermia modulates immune-related genes in various organs

We next investigated the ability of FRH to induce changes in immune-related gene expression. In livers from FRH-treated rats, we evaluated the expression of Csf3, Mif and IL10. We observed an important and immediate increase in Csf3 expression upon FRH treatment (p < .01; Figure 3(A)). This effect was significantly reversed after 24 h (p < .01). No changes in Mif expression were observed directly after FRH treatment in rat livers (Figure 3(B)), but a substantial decrease in its expression 24 h post-FRH treatment was observed. No significant changes in IL10 mRNA expression were seen (Figure 3(C)). Ifng could not be detected (data not shown).

Figure 3. Effects of FRH on the expression of (A) Csf3, (B) Mif and (C) IL10 in rat livers. mRNA expression was determined by RT-qPCR. Data are shown as the mean ± SEM of three independent experiments. Asterisks indicate significant difference between groups (**p < .01, *p < .05). NT: control animals (n = 6), FRH-t₀: samples collected directly following FRH treatment (n = 6), FRH-t₂₄: samples collected 24 h post-FRH treatment (n = 5), n: sample size per group.

We next examined spleen tissues from rats treated with FRH. We observed a significant increase in Csf3 expression following FRH treatment (p < .01; Figure 4(A)) that was abolished after 24 h (p < .001). This decrease was statistically significant compared to nontreated animals (p < .001). In contrast to Csf3, we observed a substantial decrease in Mif expression in the spleen immediately following FRH (p < .01; Figure 4(B)) which continued to decline after 24 h (p < .001). Mif expression 24 h post-FRH was indeed lower than that in nontreated rats (p < .001). A modest rise in IL10 expression directly after FRH treatment was also observed, but this effect was not statistically significant (p = .67; Figure 4(C)). After 24 h, we observed an increase in IL10 expression in the spleen compared to nontreated animals (p < .05). No changes in Ifng expression occurred in the spleen in response to FRH (Figure 4(D)). A modest increase in Ifng expression was observed after 24h, but this was not significant (p = .17).

Figure 4. Effects of FRH on the expression of (A) Csf3, (B) Mif, (C) IL10 and (D) Ifng in the spleen of rats. mRNA expression was determined by RT-qPCR. Data are shown as the mean ± SEM of three independent experiments. Asterisks indicate significant differences between groups (***p < .001, **p < .01, *p < .05). NT: control animals (n = 6), FRH-t₀: samples collected directly following FRH treatment (n = 6), FRH-t₂₄: samples collected 24 h post-FRH treatment (n = 5). n indicates the sample size per group.

Finally, we investigated changes in gene expression in the PBMCs isolated from rats. No significant differences in Csf3 expression following FRH treatment were observed (Figure 5(A)). However, a reduction of Csf3 expression was observed in PBMCs 24 h post-FRH treatment compared to the control group (p < .05). Furthermore, no changes in Mif expression post-FRH was observed (p = .22; Figure 5(B)), but after 24 h, its expression significantly declined (p < .05) in comparison to the FRH-t₀ group. We observed a substantial rise in IL10 in PBMCs following FRH treatment (p < .001; Figure 5(C)), which decreased after 24 h (p < .001). Although IL10 expression remained high after 24 h, no substantial change was observed in comparison to nontreated controls (p = .10). Looking at Ifng, a substantial growth in the PBMCs of FRH-treated rats was observed (p < .01; Figure 5(D)). This effect decreased after 24 h (p < .001), at which point its levels were comparable to the nontreated control group.

Figure 5. Effects of FRH on the expression of (A) Csf3, (B) Mif , (C) IL10 and (D) Ifng in PBMCs of rats. mRNA expression was determined by RT-qPCR. Data are the mean ± SEM of three independent experiments. Asterisks indicate significant differences between groups (***p < .001, **p < .01, *p < .05). NT: control animals (n = 6), FRH-t₀: samples collected directly following FRH treatment (n = 6), FRH-t₂₄: samples collected 24 h post-FRH treatment (n = 5). n indicates the sample size per group.

3.4. Fever-range hyperthermia affects the expression of the miRNA machinery

To identify changes in miRNA expression in FRH-treated rats, levels of key members of the miRNA machinery, including Dicer1 and Tarbp2 were determined in PBMCs, liver and spleen tissues. In PBMCs, a significant rise in Dicer1 mRNA expression was observed immediately after FRH treatment (p < .01; Figure 6(A)), which was abolished after 24 h (p < .001). Similarly, we observed a substantial growth in Tarbp2 expression in PBMCs following FRH treatment (p < .001; Figure 6(B)), with the return to physiological levels seen after 24 h (p < .001).

Figure 6. Effects of fever range hyperthermia on the expression of (A) Dicer1 and (B) Tarbp2 in PBMCs of rats. mRNA expression was assessed via RT-qPCR. Data are shown as the mean ± SEM of three independent experiments. Asterisks indicate significant difference between groups (***p < .001, **p < .01). NT: control animals (n = 6), FRH-t₀: samples collected directly after FRH treatment (n = 6), FRH-t₂₄: samples collected 24 h post-FRH treatment (n = 5), n: sample size per group.

We next measured the expression of Dicer1 and Tarbp2 in the liver as this is known to be a heat-sensitive organ. Directly following FRH treatment, we observed a significant growth in Dicer1 mRNA expression (p < .01), which declined after 24 h (p < .05; Figure 7(A)). We observed a further substantial increase in Tarbp2 expression directly following FRH treatment (p < .05; Figure 7(B)).

Figure 7. Effects of fever-range hyperthermia on the expression of (A) Dicer1 and (B) Tarbp2 in the liver of rats. mRNA expression was determined by RT-qPCR. Data are shown as the mean ± SEM of three independent experiments. Asterisks indicate significant difference between groups (**p < .01, *p < .05). NT: control animals (n = 6), FRH-t₀: samples collected directly following FRH treatment (n = 6), FRH-t₂₄: samples collected 24 h post-FRH treatment (n = 5). n indicates the sample size per group.

We next investigated Dicer1 and Tarbp2 expression in the spleen, given its key role in immune defenses. Similarly, to both liver tissue and PBMCs, we observed an important increase in the expression of Dicer1 immediately after FRH (p < .05), which returned to physiological levels after 24 h (Figure 8(A)). The levels of Tarbp2 in the spleen also raised directly following FRH (p < .05; Figure 8(B)) and remained high after 24 h, though this effect was not statistically significant (p = .16).

Figure 8. Effects of fever-range hyperthermia on the mRNA expression of (A) Dicer1 and (B) Tarbp2 in the spleen of rats. mRNA expression was determined by RT-qPCR. Data are shown as the mean ± SEM of three independent experiments. Asterisks indicate significant difference between groups (***p < .001, **p < 0.01, *p < .05). NT: control animals (n = 6), FRH-t₀: samples collected directly following FRH treatment (n = 6), FRH-t₂₄: samples collected 24 h post-FRH treatment (n = 5). n indicates the sample size per group.

3.5. Whole body hyperthermia influences miRNA-155 expression in rats

To confirm whether the effects of FRH on the miRNA machinery leads to changes in miRNA expression, we analyzed miRNA-155 levels. This molecule is well-known for promoting the pro-inflammatory response [23], thus we wanted to determine whether FRH may affect its expression. Interestingly, we found that FRH decreased the expression of miRNA-155 in rat plasma immediately following treatment (p < .001; Figure 9). This effect remained after 24 h (p < .001).

Figure 9. Effects of fever-range hyperthermia on miRNA-155 expression in the plasma of rats. miRNA expression was determined by RT-qPCR. Data are shown as the mean ± SEM. Asterisks indicate significant differences between groups (***p < .001). NT: control animals (n = 6), FRH-t₀: samples collected directly after FRH treatment (n = 6), FRH-t₂₄: samples collected 24 h post-FRH treatment (n = 5). n indicates the sample size per group.

4. Discussion

Fever is a part of the acute phase response that is manifested with an increase in body temperature and sickness symptoms such as lethargy or depression [1]. Although fever is observed in many medical conditions, the role of the heat is often ignored and antipyretics are commonly used. FRH is a simplified model of fever and a well-established medical procedure that is used in medicine. While medical devices have been designed to apply whole body hyperthermia, its use has been stalled in modern medicine. This is due to the relatively smaller number of publications and clinical trials describing the benefits of whole body hyperthermia. The literature is dominated by observational studies, with a limited understanding of the molecular mechanism(s) induced by hyperthermia [24,25].

In humans, infrared radiation has been used to achieve reproducible ranges of fever. The duration of the heat-retention phase for FRH is mostly 60 min, however, it may be prolonged up to 90 min for treatment disorders such as ankylosing spondylitis (AS), or in other fields of medicine such as rheumatology, dermatology or psychiatry [26–28]. In a particular case, such as supporting cancer treatment, even longer therapy is recommended [26].

In an effort to imitate infrared-triggered FRH, we developed a rapid method of FRH in rats. As smaller animals are more sensitive to heat because of their higher surface-to-mass ratio and the presence of fur [29,30] we decided to maintain the rats’ core body temperature at 39 °C for 60 min. Before this study, hyperthermia in animal models was usually achieved using convection models such as streams of heated air [31,32] water-baths [33–35] or a high ambient temperature in an incubator or chamber [36,37]. In addition, these models were used in research focused on application of FRH for cancer [34,35], or rheumatic disorders treatment [37], showing therapeutic potential of FRH in these disorders.

There are a few differences between the types of hyperthermia models. In convection models of FRH skin and the surface of the body are heated first, and then, heat is transferred by blood to the whole body. This method of whole body hyperthermia induction requires more time than infrared-induced FRH due to the prolonged heat-retention phase. Unlike the convection model of FRH, infrared radiation penetrates the deep layers of skin and then heat is transferred to the core of the body through blood circulation. The skin heats up less therefore this method allows for limitation of the risk of skin burns [38,39]. Importantly, in our model based on infrared radiation, we achieved fever-range hyperthermia within minutes, while other methods need approximately twice a longer time [34,35].

The main limitation of various models of hyperthermia (including ours) is the necessity to anesthetize rats, if stress prevention is needed. It is known that rats in response to various stressors (e.g., moving cages, noise and light) develop a stress-induced increase in body temperature [40]. These kinds of stressors affect the secretion of neuroendocrine mediators, exposing immune cells to altered signals and interactions [40,41]. On the other hand, it should be kept in mind that anesthesia induces vasodilation that weakens thermoregulatory defense mechanisms [42,43]. Therefore, before choosing an appropriate model of FRH-induction, a wide spectrum of potential risks should be considered, including the way of core temperature measurements.

Having refined a model replicating FRH in humans, we assessed changes in the number of leukocytes in the blood. As other authors, who investigated different models of hyperthermia, observed changes in the immune system after 24 h including the number of white blood cells count and level of various cytokines [27,44], we wanted to check whether a similar effect is observed in our model. Indeed, we found that FRH provoked a temporary decrease in the total number of WBCs that returned to initial levels after 24 h. The observed changes in the number of WBCs were due to a decrease in the number of lymphocytes. In agreement with these data, a decreased number of lymphocytes directly following FRH treatment was observed in patients with advanced solid tumors [44]. As FRH is not cytotoxic for WBCs [5,45], hyperthermia likely enhances the diapedesis of lymphocytes and intensifies immune surveillance.

Unlike lymphocytes, we observed a significant increase in granulocyte number following FRH. This suggested that FRH may enhance granulocytopoiesis. To verify this hypothesis, we examined the expression of G-CSF (encoded by the Csf3 gene), a key growth factor that regulates granulocytopoiesis, induces the proliferation of granulocytic precursors and promotes the release of mature cells from the bone marrow into the blood [46]. The analysis of these genes revealed increased expression immediately after FRH in all examined organs. Of note, Csf3 expression was most pronounced (100-fold increase vs. control group) in the liver. As Piscaglia et al. [47] highlighted the ability of G-CSF to induce hepatic regeneration by increasing the migration of bone marrow progenitors to the liver our results suggest that FRH could be beneficial for the treatment of liver injuries by inducing G-CSF expression.

We further investigated the effects of FRH on the count of monocytes. As G-CSF is a key regulator of granulocytopoiesis and monocytopoiesis [48], we anticipated downstream effects of FRH on monocyte number. Surprisingly, we observed a modest but nonsignificant change in the number of monocytes in blood samples. We additionally evaluated the expression of Mif, and observed decreased levels in all examined organs after 24 h of FRH. Despite its obvious role in the regulation of the migration of macrophages, MIF regulates the expression of pro-inflammatory cytokines. Furthermore, it is a key regulator of the innate immune system, most notably the inflammatory response to microbial infection [9]. These findings suggest that FRH acts as an immunomodulatory factor responsible for the maintenance of immune homeostasis. FRH has been shown to influence the expression of anti-inflammatory cytokines including IL-10 [12]. Consistent with these studies, we observed that its expression in PBMCs and spleen significantly increased following FRH. Thus, we found that FRH regulates both pro-inflammatory and anti-inflammatory factors.

Previous studies have highlighted an interplay between G-CSF and IL-10. Shaklee et al. [49] found that G-CSF increases LPS-induced IL-10 expression in the spleen. Consistent with these studies, we observed a significant increase in spleen G-CSF mRNA levels directly after FRH treatment, followed by an increase in the expression of IL-10 after 24 h. Zauner et al. observed, that hyperthermia treatment induced expression of anti-inflammatory IL-10 in patients with AS and the control group. However, this increase was earlier, higher and more sustained in AS patients [27]. This increase is in line with our observations, thus FRH seems to be useful for arthritis treatment. Borges et al. explained that increase in IL-10 level may result from HSP-70 treatment. They observed, that extracellular HSP-70 decrease pro-inflammatory cytokines level due to increased IL-10 production in bone marrow-derived murine dendritic cells [50].

In our study, IFN-γ mRNA levels in rats also increased in PBMCs, but only modestly increased in the spleen. It is well-known that IFN-γ is a molecule synthesized in response to viral attack to limit viral infection [10]. In experiments designed to induce its production in lymphoid cells, the activity of mitogens or cytokines are needed [51]. Until recently, the ability of hyperthermia to induce IFN-γ was restricted to simultaneous stimulation with other factors. Zhu et al. [2] found that local hyperthermia-induced temperature dependent increases in IFN-γ in HPV-infected tissues, while Downing et al. [52] reported increased IFN-γ expression in PHA-stimulated lymphocytes isolated from patients treated with FRH at 39 °C. Here, we observed that FRH alone could stimulate IFN-γ, which was in accordance with Mace et al. [53], who showed that FRH induces temperature-dependent IFN-γ expression in murine CD8+ T cells in vitro.

Having observed the effect of FRH on cytokine production, we next investigated the potential role of miRNAs. miRNAs have emerged as key regulators of biological processes including cell development, differentiation and homeostasis [17,54]. Importantly, various miRNA molecules regulate the expression of G-CSF [13], MIF [14], IFN-γ [15] and IL-10 [16]. We, therefore, investigated the influence of heat on the expression of the machinery required for miRNA processing. Mature miRNA duplexes are recruited for the RNA-induced silencing complex (RISC). The miRNA machinery includes e.g. DICER1 (RNase III family member) and transactivation response RNA-Binding Protein (TARBP2 also known as TRBP) [55]. In rats, we observed altered expression of Dicer1 and Tarbp2 in all organs examined. These data are consistent with in vitro studies highlighting the ability of heat to induce the expression of DICER1 [56,57]. Increasing data suggest that other molecules involved in miRNA machinery such as argonaute2 protein (Ago2), may be modulated by heat shock proteins (HSPs) [58,59]. These results reveal that the regulation of miRNA machinery is even more complicated. Therefore, further functional analysis is required. However, to the best of our knowledge, no study has considered heat-induced Tarbp2 expression. Hence, our results provide new insight into the role of miRNA machinery in response to FRH.

We next investigated whether FRH can influence the expression of miRNA-155, a known regulator of both innate and adaptive immune responses [23]. We found that FRH led to a significant decrease in miRNA-155 expression in rat plasma, further highlighting its ability to promote anti-inflammatory responses. This ability of FRH may be beneficial for a range of disease pathologies involving chronic inflammation, including rheumatoid arthritis. Indeed, the therapeutic benefits of FRH in controlling the progression of arthritis in clinically relevant mouse models are comparable to methotrexate. Heat treatment has been shown to increase IL-10 production in inflamed joints [37], though the molecular mechanisms of these effects have not been investigated. We observed an FRH-dependent decrease in MIF expression that regulates the expression of pro-inflammatory cytokines such as IL-6 and TNF-α [60]. Furthermore, elevated levels of miRNA-155 have been reported in the synovial tissue of rheumatoid arthritis patients, which precedes with the release of pro-inflammatory cytokines by fibroblast‑like synoviocytes [61]. We observed that FRH induces a decrease in miRNA-155, downregulates MIF and upregulates IL-10, which may be beneficial for chronic disease. This requires further investigation, as in vitro findings by Li et al. suggest [62] that the increased expression of miRNA-155 in heat-treated microglial cells increases the expression of pro-inflammatory molecules.

In summary, we show that FRH significantly influences WBC numbers and immune-related gene expression in an array of organs. Our data support FRH as an immunomodulator that influences key anti-inflammatory factors including IL-10, as well as pro-inflammatory molecules such as MIF and IFN-γ. Of note, upregulation of G-CSF, which is considered to display a dual role in inflammation [63], was also observed. Regarding changes in miRNA machinery expression and miRNA-155 itself, we hypothesize that all the changes described above may occur through the regulation of miRNAs.

Ethical approval

The animal study protocol was approved by the local Bioethical Committee for Animal Care of Bydgoszcz University of Science and Technology at the Faculty of Animal Breeding and Biology (permission no. 49/2020, date of approval 24.01.2020).

Author contributions

Conceptualization, H.M.K. and S.W.; methodology, H.M.K., B.M., J.S. and T.J.; software, H.M.K. and B.M.; validation, T.J and J.S.; formal analysis, T.J. and J.S.; investigation, H.M.K. and B.M.; resources, H.M.K., S.W. and A.D.; data curation, H.M.K. and B.M.; writing—original draft preparation, H.M.K.; writing—review and editing, T.J. and S.W.; visualization, H.M.K. and J.S.; supervision, S.W. and A.D.; project administration, H.M.K. and S.W.; funding acquisition, S.W. and A.D.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article. The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Additional information

Funding

This study was supported by Project no POWR.03.05.00-00-Z302/17 “Universitas Copernicana Thoruniensis in Futuro” (2018-2022) co-financed by the European Social Fund—the Operational Programme Knowledge Education Development. Module 5. Interdisciplinary PhD School “Academia Copernicana” and the Polish Minister of Science and Higher Education under the program “Regional Initiative of Excellence” in 2019–2023 [Grant No. 008/RID/2018/19].