key: cord-0805329-75qwz4y6
authors: de Queiroz Rodrigues, Maria Imaculada; de Lima Martins, Joyce Ohana; de Barros Silva, Paulo Goberlânio; Ferreira Júnior, Antônio Ernando Carlos; Lima Verde, Maria Elisa Quezado; Sousa, Fabrício Bitú; Mota, Mário Rogério Lima; Negreiros Nunes Alves, Ana Paula
title: Tocilizumab, a potent interleukin-6 receptor inhibitor, decreases bone resorption and increases the rate of bacterial infection after tooth extraction in rats
date: 2020-08-15
journal: J Oral Maxillofac Surg
DOI: 10.1016/j.joms.2020.08.012
sha: fe8c1413d57545aabdfe09f9d69a4d3a7652da4d
doc_id: 805329
cord_uid: 75qwz4y6

PURPOSE: Our objective was to evaluate the influence of pre-treatment with tocilizumab (TCZ) in bone healing after tooth extraction in rats. METHODS: Wistar male rats were equally divided into sham (i.e., non-operated), saline (both treated with 0.1 ml/kg saline), and six TCZ groups treated with 1, 2, 4, 8, 16, and 32 mg/kg TCZ (TCZ1 to TCZ32, respectively). Twenty-four hours after administration of vehicle or TCZ, exodontia of the first lower left molar was performed, and the animals were euthanized three days later for hematological analysis and organ (liver, spleen, and kidney mass indexes, and histological evaluation), gingiva (myeloperoxidase [MPO] assay), and mandible (radiographic, histomorphometric analysis, and IL-6 immunostaining) evaluation. Analysis of variance/Bonferroni test (statistical significance, p<0.05) was performed using GraphPad Prism version 5.0 (GraphPad Inc, San Diego, CA, USA). RESULTS: There was no difference in radiographic results; however, leukopenia (p=0.039) and neutropenia (p<0.001) were statistically significant in the TCZ16 and TCZ32 groups. Weight loss (p<0.001) and reduced liver index (p=0.001) were significantly dose dependent; however, no histological alterations were observed in the other organs. Osteoclast counts were reduced in groups TCZ4 to TCZ32 (p<0.001), and IL-6 immunostaining increased in the TCZ8 to TCZ32 groups (p<0.001). Alveolar infection rates increased in groups TCZ4 to TCZ32 (p<0.001), and MPO had biphasic response, exhibiting a reduction in groups TCZ2 and TCZ4, and an increase in group TCZ32 (p=0.004). CONCLUSION: TCZ-induced immunosuppression led to a reduction in osteoclast function, an increase in alveolar infection, and compensatory neutrophil infiltration.

Monoclonal antibodies are a class of drugs widely used to treat autoimmune diseases and some cancers, as well as for immune control of infections occurring as side effects. The mechanism involves the blockage of cytokines or receptors that inhibit the natural course of some diseases. 1 This therapeutic approach has improved the health of some patients with chronic local and systemic diseases. 2 A humanized anti-interleukin (IL)-6 receptor monoclonal antibody, tocilizumab (TCZ), has recently been authorized for use in humans. TCZ binds reversibly to the IL-6 receptor (IL-6R), blocking Janus kinase/signal transducers and activators of transcription (JAK/STAT) activation and the subsequent overproduction of kidney cancers. 4 Recently, TCZ has been used as palliative treatment for oral squamous cell carcinomas 5,6 and multi-drug-resistant anemia 7,8 ; thus, its range of therapeutic applications has increased.

Furthermore, TCZ has been used, with good results, in the treatment of patients with coronavirus disease 2019 , caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). 9 In these conditions, a large number of critically ill patients experience a "cytokine storm" caused by the overproduction of proinflammatory cytokines, especially IL-6. This storm contributes to increased disease severity and poorer prognosis, and TCZ contributes to improvement of symptoms by reducing the inflammation associated with this IL-6 storm. 10, 11 Blockage of IL-6R reduces the overexpression of several inflammatory chemical mediators that are important in osteoclastogenesis, 4 interfering with bone remodeling. 12 After exodontia, there is a rapid and natural increase in the levels of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), IL-1β, and IL-6, leading to the production of receptor activator of nuclear factor kappa-B ligand (RANKL) and bone loss. 4, 13 Despite pathological bone loss being attenuated, 14, 15 the interruption of inflammatory pathways can delay bone remodeling. 16 After exodontia in dental-exposed alveolus, delayed bone remodeling can heighten infection risk due to the rich oral microbiota 17 and the blockage of neutrophil migration and macrophage activation by the blockage of IL-6 activity. 18 Based on the wide therapeutic potential of TCZ and its relevance in clinical dental procedures, the objective of the present study was to evaluate the effect of pretreatment with TCZ on bone remodeling in rats subjected to tooth extraction. J o u r n a l P r e -p r o o f of "multiples of two", as previously described. 19 Exodontia was not performed in the J o u r n a l P r e -p r o o f sham group, and rats in both the saline and TCZ groups underwent extraction of the first left lower molar.

Twenty-four hours after saline or TCZ injection (day 0), the animals were weighed (initial weight), anesthetized with an intraperitoneal injection of a freshly prepared mixture of ketamine (90 mg/kg) and xylazine (10 mg/kg) and subjected to tooth extraction (Hollemback 3s spatula) or sham surgery in a blinded manner. On the day exodontia was performed, 2 mL of blood (retro-orbital plexus) was collected to count leukocytes (myeloid and lymphoid cells [initial leukocyte count]).

After exodontia, the teeth were dried on absorbent paper and weighed using a precision balance. Additionally, an operator blinded to the group assignments recorded the number of root fractures. 20 Three days after surgery (day 3), the animals were weighed and anesthetized, and 2 mL of blood (retro-orbital plexus) was collected to count leukocytes (myeloid and lymphoid cells) (Sysmex KX 21N, Roche). The animals were euthanized using ketamine (270 mg/kg) and xylazine (30 mg/kg) overdose, and alveolar mucosa, alveolar bone, kidneys, spleen, and liver samples were collected.

Weight and leukocyte count evaluations were performed by dividing the final value by the initial value multiplied by 100%.

On the day the animals were euthanized, the liver, spleen, and kidneys were removed and weighed to verify the organ index. The organ index was calculated by dividing the weight of the organ by the final weight of the rat multiplied by 100%.

These organs and alveolar bone were then fixed in 10% neutral formalin solution for macroscopic and microscopic evaluation.

After the organs were subjected to macroscopic analysis, they were histologically processed and embedded in paraffin blocks. Sections (4-μm thick) were cut using a semi-automatic microtome and stained with hematoxylin and eosin for examination under a conventional light microscope (magnification 40×, 100×, and 400×) and histological analysis of the organs was based on the presence and extension of lesions. 20

The mandibles of all groups underwent radiography using a conventional X- After randomization, an operator blinded to the group assignments manually measured the radiolucent area in triplicate using a freehand selection approach. Using the measure command, the area of the demarcated radiolucent region was measured. The average value of three measurements was designated as the sample unit. 20 J o u r n a l P r e -p r o o f

The mandibles were fixed in a 10% neutral formalin solution and decalcified (10% EDTA, pH 7.3, NaOH, PA) for 30 days and maintained in suspension.

After the hemi-mandibles were histologically processed and embedded in paraffin blocks, sections (4-µm thick) were cut using a semi-automatic microtome and stained with hematoxylin and eosin for examination under a conventional light microscope (magnification 400×).

Ten micro-fields were selected for micrographs using a Leica DFC295 microscope coupled to a Leica DM 2000 microscope with LAS software (Leica, Heerbrugg, Switzerland). The images were exported and analyzed using ImageJ software. The cell counter command was used to count osteoclasts. To minimize observation bias, the slides were randomized, and the investigator was blinded to the group assignments. The sum of 10 micrographs was considered the sample unit for statistical analysis. 21, 22 

The referent area of the alveolus was marked on a slide with a permanent brush to select the area for the tissue micro array procedure. The slide was paired with a paraffin block and a tissue microarrayer (Quick-Ray Unitma Co. Ltd., Seoul, Korea) was used to punch a 2-mm diameter sample of material from the paraffin block. The sample was transferred to two receptor paraffin blocks with capacity for 36 samples each. For the immunohistochemical assay, sections (4-μm thick) were cut using a semiautomatic microtome and mounted on silanized slides.

After deparaffinization and rehydration, antigen retrieval was performed by heating in citrate solution (pH 6.0). After reaching room temperature, the slides were blocked in peroxidase with 3% hydrogen peroxide (H2O2) and diluted in phosphatebuffered saline for 30 min. After being blocked with albumin for 30 min, the slides were incubated with IL-6 antibody (1:300 dilution; Abcam, Cambridge, United Kingdom).

Universal Immune-peroxidase Polymer (Histofine, Nicherei Biosciences

Inc., Tokyo, Japan) for 30 min was used as the secondary antibody and 5,5diaminobenzidine tetrahydrochloride (DAB; Dako, Dopenhage, Denmark) was used to identify positive cells. Harris hematoxylin was used for counter staining.

The same method of osteoclast counting was performed to obtain photomicrographs of IL-6 reactions. The cell counter command was used to count IL-6 cytoplasm-positive cells. To minimize observation bias, the slides were randomized, and the investigator was blinded to the group assignments. The sum of 10 micrographs was considered a sample unit for statistical analysis. 21, 22 

A myeloperoxidase (MPO) quantification assay is broadly used as a marker for myeloid cell infiltration into tissues, mainly neutrophils. Following euthanasia, a specimen of the alveolar mucosa was collected, weighed, and freeze-dried at -80°C until 

The Shapiro-Wilk test was used to assess the normality of data distribution.

Data are expressed as mean ± standard error of the mean (SEM) or absolute or relative frequency. Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, Inc., San Diego, CA, USA). One-way ANOVA/Bonferroni or chisquared tests were used to compare the groups. The level of statistical significance was set at 5% (i.e., p < 0.05).

When the weight of each tooth was analyzed, we verified that there was no statistically significant difference between the non-operated sham group and the other groups (p = 0.602). The number of radicular fractures did not differ between the control and TCZ groups (p = 0.910) in the experimental groups (Table 1) .

There were no significant differences in the variation in leukocyte count between the sham group, control group and rats in groups TCZ1, TCZ2, and TCZ4.

However, rats in groups TCZ8, TCZ16, and TCZ32 exhibited a significant decrease in the total number of total leukocytes (p = 0.039) from day 0 to day 3 (Table 1) .

Although there were no differences in variation of lymphoid cell counts among the groups (p = 0.310), the TCZ16 and TCZ32 groups exhibited significant decreases in the variation of myeloid cell counts without differences among the sham, control, TCZ1, TCZ2, TCZ4, and TCZ8 groups ( Table 1) .

Weight loss was greater in the TCZ16 and TCZ32 groups than in the other groups (p < 0.001) ( Table 1 ). The spleen (p = 0.972) and kidney (p = 0.420) indexes did not exhibit significant differences; however, there was a significant reduction in liver index in animals in the TCZ16 and TCZ32 groups compared with the other groups (p = 0.001) ( Table 1) .

Histological parameters, including renal, splenic and hepatic toxicity, did not demonstrate significant variation among the eight experimental groups (Supplementary tables 1 to 3).

All animals that underwent exodontia of the first left inferior molar exhibited a radiolucent dental alveolus surrounded by a thin radiopaque line. The radiolucent area of tooth extractions did not differ among the saline (1376 ± 130 pixels), TCZ1 (1401 ± 92 pixels), TCZ2 (1255 ± 71 pixels), TCZ4 (1247 ± 146 pixels), TCZ8

(1282 ± 107 pixels), TCZ16 (1413 ± 128 pixels), and TCZ32 (pixels) groups (p = 0.867) (Figure 1 ).

The sham group exhibited root surfaces and periodontal ligaments without morphological alterations. The control group exhibited an exposed dental alveolus with groups exhibited a significant reduction in osteoclast count (p < 0.001) (Figure 2 and 3).

The number of rats exhibiting bacterial colonies suggestive of Actinomyces was significantly higher in the TCZ4, TCZ8, TCZ16, and TCZ32 (100%) groups than in the other groups (sham, 0%; control, 25%; TCZ1, 17%; and TCZ2, 40%) (p < 0.001).

These data suggest that the increase in the number of bacterial colonies was dose dependent.

MPO activity in the sham group (7.6 ± 0.9) was significantly lower than that of the control group (36.5 ± 7.1). There was no difference in MPO activity between the control group and TCZ1 (36.5 ± 7.1). The TCZ2 (10.4 ± 0.1) and TCZ4 (9.0 ± 0.5) groups exhibited a significant decrease in MPO activity, and the TCZ32 group (66.2 ± 29.3) exhibited a significant increase (66.2 ± 29.3) (p = 0.004) (Figure 4 ).

The mean number of IL-6-positive cells in the sham group (48.5 ± 17.0) was significantly lower than that of the control group (333.5 ± 93.8). There was no difference in MPO activity between the control group and TCZ1 (320.5 ± 98.3), TCZ2

(280.3 ± 56.8), and TCZ4 (650.7 ± 182.9) groups; however, rats in the TCZ8 (1510.0 ± 294.2), TCZ16 (1623.0 ± 85.9), and TCZ32 (1474 ± 128.7) groups exhibited a higher number of IL-6-positive cells than the saline group (p < 0.001) (Figure 2 and 3 ).

TCZ is a monoclonal antibody that binds to the IL-6R, inhibiting its activation and blocking the activation of IL-6. Thus, this mechanism is vital in controlling diseases characterized by the overproduction of IL-6. 24 Because this agent has a wide range of therapeutic applications, we studied initial bone remodeling posttooth extraction in rats pretreated with TCZ. We evaluated the tooth alveolus three days post-exodontia because this is the day with the highest number of inflammatory cells. 23 So, the maximum impact of IL-6R blockage in inflammatory cell migration would be observed in tooth alveolus in this time.

IL-6 is an important cytokine related to some physiological processes.

Although controlling IL-6 is considered to be indispensable in the treatment of some diseases, including periodontitis, 25 its total or partial blockage can impact physiological functions. 26 Leukocyte counts in the groups treated with high doses of TCZ exhibited significant leukopenia that was myeloid cell dependent. IL-6 is indispensable in the J o u r n a l P r e -p r o o f late phase of myeloid differentiation; 27 however, its effect on lymphoid cells appears to be more qualitative (modulation of the T-helper cell immune profile response) than quantitative. 28 This explains the more intense effect of TCZ in cells with a myeloid than lymphoid lineage. Additionally, this could explain the weight loss in the two high-dose groups because suppression of the medulla is often accompanied by intense cachexia. 29 Histological analysis revealed that TCZ reduced osteoclast counts in the therapeutic doses (i.e., TCZ4 and TCZ8) and overdoses (i.e., TCZ16 and TCZ32) in alveolar bone. Immediately after exodontia, there is an increase in the levels of some pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, which lead to monocyte recruitment and osteoclast differentiation. 30 IL-8 production in response to bacterial byproducts is independent of TNF-α, IL-1β, and IL-6. In the severe absence of IL-6 (i.e., TCZ32 group), macrophage activity was engaged, such as osteoclast function; therefore, compensatory IL-8 overexpression can lead to a high level of neutrophil infiltration. 34 Neutrophils produce high levels of prostaglandin E2, 37 which inhibit MCP-1 production by reducing infiltration of progenitor osteoclast cells 38 and lead to tissue damage 39 such as osteomyelitis. 40 There were no significant alterations to the kidneys or spleen; however, TCZ caused significant hepatic damage. Hepatotoxicity is a limiting factor to TCZ treatment in patients with rheumatoid arthritis, 41 which has been described in case reports of severe hepatitis after TCZ use. 42 It is probable that the indispensable axis involving IL-6 and hepatocyte growth factor is partly responsible for this toxicity. 42, 43 Although TCZ does not directly inhibit the IL-6R in rodents, 44 some experimental models have demonstrated its benefits in rats. 19, 45 In addition, IL-6R is mainly found on hepatocytes, neutrophils, monocytes, and LTCD4, 46 all of which were altered in this experimental model.

Inevitably, there will be an increase in the use of TCZ due to the current COVID-19 pandemic. A clinical human dose of TCZ (8 mg/kg) has a long half-life (> 4 weeks). 9 In patients with COVID-19, the administration of TCZ quickly improves respiratory symptoms, and further doses are necessary for disease control. 11 Results of our study can help in understanding why some COVID-19 patients worsen after TCZ treatment. With high suppression of the IL-6 pathway, secondary bacterial infections can activate non-canonical pathways, leading to the overexpression of both IL-6 and J o u r n a l P r e -p r o o f other cytokines. 10 Although this was not the focus of our study, indications for TCZ use to control inflammatory processes will increase significantly in the future, and dentistry will need to prepare for the adverse effects of this monoclonal antibody even in a short period of time.. In rats IL-6 -/there is an increase in inflammatory alveolar bone destruction, because other cytokines like IL-1α increase compensatively. 47 So, the suppression of IL-6 is likely, even at low doses, to have significant adverse effects on the alveolar bone.

In presented as mean ± standard error; n = 6/group). Original magnification ×400.

J o u r n a l P r e -p r o o f Tables   Table 1 . Surgical difficulty and systemic parameters of toxicity in rats that underwent exodontia of the first lower molar and treated with varying doses of tocilizumab (TCZ).

*p<0.05 versus sham group; † p<0.05 versus saline group (ANOVA/Bonferroni, data presented as mean ± standard error; n=6/group). Weight variation calculated as ratio of final/initial weight. Organ weight calculated as the ratio between the weight of each organ and the weight of the rat on the day of euthanization. Absence 0/6 0/6 0/6 0/4 0/5 0/6 0/6 0/6 1,000 Presence 6/6 6/6 6/6 4/4 5/5 6/6 6/6 6/6 Moderate 0/6 0/6 0/6 0/4 0/5 0/6 0/6 0/6 Bowman capsule space reduction Absence 1/6 6/6 4/6 1/4 3/5 3/6 4/6 4/6 0,137 Discrete 5/6 0/6 2/6 3/4 2/5 3/6 2/6 2/6 Tubular epithelial cell swelling Absence 0/6 0/6 0/6 0/4 0/5 0/6 0/6 0/6 0,274 Discrete 4/6 6/6 6/6 3/4 4/5 5/6 6/6 6/6 Moderate 2/6 0/6 0/6 1/4 1/5 1/6 0/6 0/6 Tubular epithelial cell vacuolation Absence 0/6 2/6 0/6 0/4 2/5 0/6 0/6 2/6 0,329 Discrete 5/6 4/6 6/6 4/4 3/5 6/6 5/6 3/6 Moderate 0/6 0/6 0/6 0/4 0/5 0/6 1/6 1/6 Recent tubular and interstitial hemorrhaging Absence 0/6 0/6 0/6 0/4 0/5 0/6 0/6 0/6 0,649 Discrete 4/6 3/6 2/6 1/4 3/5 3/6 2/6 3/6 Moderate 2/6 3/6 4/6 3/4 2/5 3/6 4/6 3/6 Nephrotoxic necrosis Absence 5/6 6/6 6/6 4/4 5/5 6/6 6/6 6/6 0,649 Presence 1/6 0/6 0/6 0/4 0/5 0/6 0/6 0/6 Inflammatory infiltrate Absence 0/6 0/6 0/6 0/4 0/5 0/6 0/6 0/6 0,324 Discrete 4/6 5/6 6/6 4/4 5/5 4/6 6/6 6/6 Moderate 1/6 1/6 0/6 0/6 0/6 2/6 0/6 0/6 Hyaline-Cylinder Absence 0/6 0/6 0/6 0/4 0/5 0/6 1/6 1/6 0,569 Discrete 6/6 6/6 6/6 4/4 5/5 6/6 5/6 5/6 *p<0.05, chi-square test. Data expressed as absolute frequency/total.

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Absence 3/6 3/6 2/6 1/4 2/5 1/6 2/6 0/6 0,711 Presence 3/6 3/6 4/6 3/4 3/5 5/6 3/6 5/6 Moderate 0/6 0/6 0/6 0/4 0/5 0/6 1/6 1/6 Hepatocyte swelling Absence 4/6 4/6 3/6 1/4 2/5 3/6 1/6 0/6 0,313 Discrete 2/6 2/6 3/6 1/4 2/5 2/6 3/6 5/6 Moderate 0/6 0/6 0/6 2/4 1/5 1/6 2/6 1/6 Sinusoidal hemorrhage Absence 1/6 1/6 2/6 2/4 1/5 1/6 0/6 0/6 0,654 Discrete 4/6 4/6 3/6 1/4 1/5 3/6 3/6 2/6 Moderate 1/6 1/6 1/6 1/4 3/5 2/6 3/6 3/6 Intense 0/6 0/6 0/6 0/4 0/5 0/6 0/6 0/6 Inflammatory focus Absence 3/6 2/6 1/6 0/4 0/5 0/6 1/6 4/6 0,434 Discrete 3/6 4/6 4/6 2/4 2/5 3/6 1/6 1/6 Moderate 0/6 0/6 1/6 2/4 3/5 3/6 3/6 1/6 Intense 0/6 0/6 0/6 0/4 0/5 0/6 1/6 0/6 J o u r n a l P r e -p r o o f Supplementary