TLR2 Limits Tongue Tumorigenesis By Regulating The Differentiation And Invasion Of M2 Macrophages

Inflammation is closely related to oral tongue squamous cell carcinoma?OTSCC?.However,its mechanism is still obscure.Toll like receptor 2?TLR2?plays an important role in oral chronic inflammatory diseases,but the role of TLR2 in OTSCC is unclear.Here,we used 4-nitroquinoline1-oxide?4-NQQ?to induce a tongue cancer model in TLR2-/-and wild type?WT?mice.Histological results demonstrated that TLR2 played a protective role in the development and progression of OTSCC.The TLR2-/-mice showed a more aggressive tongue cancer development that was closely associated with the alternative activated?M2?macrophage abnormality.In vitro,we found that monocytes from TLR2-/-mice were more easily induced to M2 macrophages than WT mice.According to the result of Immunohistochemical analysis,the quantity of M2 macrophages under the tongue epithelium in TLR2 deficient mice was more than in the WT mice,and the number of M2 macrophage abnormalities accelerated the development of the tumor.Overall,our findings showed that TLR2 deficiency accelerated OTSCC development by manipulating M2 macrophage differentiation and infiltration.These findings provide a new idea for treating tongue cancer using effective immunotherapies through TLR2.Background and ObjectiveAbout 2%of newly diagnosed cancers in the world each year are oral cancer.Oral tongue squamous cell carcinoma?OTSCC?is the most frequently observed type of cancer in the oral cavity,accounting for around 40%of oral cancers^[1,2].However,the tumorigenesis of the tongue and the mechanism of development are unclear.Despite the fact that many new multimodal therapies have been put forward to combat OTSCC over the past decades,the five-year survival rate has not been outstanding improved,remaining at about fifty percent^[3,4].Thus,it is important to clarify the molecular mechanism of tongue tumorigenesis and develop more effective therapies.An intertwined correlation is widely confirmed between OTSCC and inflammation^[5,20].Toll-like receptors?TLRs?,described as the initiation of inflammation,are a family of receptors that recognize various PAMPs and DAMPs,which play key roles in both the innate and adaptive immune systems^[6,8].Since TLRs were firstly discovered,a total 10 TLRs?TLR1 to TLR10?and three pseudogenes?TLR11 to TLR13?were found in humans and mice^[8].These receptors play vital roles in the oral immune defense system by detecting different microbial molecular structures and triggering innate immune responses to maintain homeostasis^[6].They are not only related to tumor-related inflammation by identifying different ligands but are also expressed in many kinds of cancerous cells that are closely associating with tumor-induced immunosuppression^[9].TLR2 is a hot topic of the TLR family in tumor research,since it is widely expressed on the surface of monocytes,macrophages,DCs and PMNs,and it is extensively involved in tumorigenesis^[11].The relationship between TLR2 and tumorigenesis has been reported in many studies^[10].TLR2 limited the development of hepatocellular carcinoma as shown by Lin.In addition,Lin reported a decrease in autophagy and apoptotic-associated cell death in TLR2-/-mouse livers and a decrease in the liver infiltrating macrophage number^[12].Li found that TLR2 deficiency may lead to activation of caspase-8 and the IL-18 axis,which drives MDSC accumulation in the livers of TLR2-deficience mice,and the accumulation of MDSCs leaded to powerful suppression of antitumor immunity during hepatocarcinogenesis^[13].However,Shi reported that downregulation of TLR2 inhibits the bioactivity of HCC cell lines^[14].As for colorectal cancer,Lowe reported that TLR2 plays a protective role against the development of colitis-induced cancer in which TLR2 deficiency led to inflammatory growth signals and a predisposition to accelerate neoplastic growth^[15].Scheeren found that TLR2 or MYD88 deficiency in the intestinal epithelium led to the reduction of spontaneous tumor development in mice^[16].In gastric cancer and gastric epithelium,TLR2 is highly expressed by STAT3 pathway regulation and promotes the progression of gastric cancer,and the targeting of TLR2 alleviates gastric tumorigenesis in animal models^[17].As for human OTSCC,TLR2 is highly expressed in each stage and in various types of OTSCC and is closely associated with the development,progression and invasiveness of OTSCC^[18,19].In summary,TLR2plays an important role in tumor progression and is closely related to MDSCs and macrophages,but its role in tongue cancer is unclear.In this article,we used 4-nitroquinoline1-oxide?4-NQQ?to induce WT and TLR2-/-mouse tongue cancer,concluded that TLR2 deficiency promotes the development of tongue cancer.We also observed changes in the alternative activated?M2?macrophages during tumor formation in the two groups.Our results showed that TLR2 deficiency made M2 macrophages more likely to infiltrate into the tumor microenvironment,which accelerated the development of tongue cancer in TLR2-deficient mice.In vitro,we found that monocytes from TLR2-/-mice were more easily induced to M2 macrophages than WT mice.MethodsExperiment 1:All of the animals were maintained in the animal facility of Nanjing Medical University under specific pathogen-free conditions and were used at6�8 weeks of age.Comparisons between the experiments were divided into four groups including the WT treated group,the WT control group,the TLR2-/-treated group,and the TLR2-/-control group,where each group contained 10 mice.The WT and TLR2 treated group mice were feed water with 4-NQQ?0.004%?until the scheduled sacrifice time,which was 8 weeks,12 weeks,16 weeks,20 weeks and 24weeks,and the control groups were wild type mice that were sacrificed at the indicated time points.The mice were killed at the indicated time points,and the tongue was removed,soaked in the 4%paraformaldehyde,dehydrated,paraffin-embedded,paraffin sectioned?0.4um?and stained for H&E.Experiment 2:Envision two-step immunohistochemical staining was performed using CD206?purchased from R&D?to label M2 macrophages.For each immunohistochemical section,10 fields were randomly selected using a 400X microscope objective,and the number of positively stained cells?M2 macrophages?was counted for the statistical analysis.In vitro,we extracted TLR2-/-and WT mouse mononuclear cells from femur bone marrow to induction of M2 macrophages,observed the difference between the two groups.ResultsExperiment 1:The mice in the control groups had no abnormalities except aging changes.As for treatment groups,the development of tongue cancer in TLR2-/-group mice was faster WT group mice,either from the overall state of the mouse or from the appearance of the tongues during the entire induction experiment.The pathological analysis results were as follows:The tongue epithelium showed no obvious changes in the WT and TLR2-/-control groups at each time point?Fig.3 a?.As for the treatment groups,there were no significant changes in the tongue epithelium of the WT group of mice at 8 weeks,but in the TLR2-/-group,four mice?40%?had mild atypical hyperplasia?P=0.029?.At 12 weeks,the WT group had seven mice?70%?with mild atypical hyperplasia,and four of the TLR2-/-mice?40%?had mild atypical hyperplasia and six?60%?had moderate atypical hyperplasia?P=0.002?.At 16 weeks,in the WT group,ten mice showed 100%mild atypical hyperplasia,and in the TLR2-/-group,two mice?20%?had mild atypical hyperplasia,seven?70%?had moderate atypical hyperplasia,and one?10%?mouse had severe and carcinoma in situ?P=0.000?.At 20 weeks,three mice?33.33%?in the WT group had mild atypical hyperplasia,four?44.44%?had moderate atypical hyperplasia,and two?22.22%?had severe atypical hyperplasia and carcinoma in situ,while two mice?20%?in the TLR2-/-grope had moderate atypical hyperplasia and eight mice?80%?had severe atypical hyperplasia and carcinoma in situ?P=0.009?.At 24 weeks,two of the WT mice?20%?had mild atypical hyperplasia,six?60%?had severe atypical hyperplasia,one mouse?10%?had severe and in situ cancer,and one?10%?had invasive carcinoma,but most of the TLR2-/-mice developed advanced lesions,which meant that five mice?62.5%?had severe atypical hyperplasia and carcinoma in situ,two mice?25%?had invasive carcinoma,and only one mouse?12.5%?had mild atypical hyperplasia?P=0.018?Experiment 2:The expression of the M2 macrophage marker CD206 was stained as membrane-bound and mainly presented a spindle-shape,and some cells showed a round shape.Exemplary fields of view showed that M2 macrophages were mainly in subepithelial basement membrane sites,and a small portion of M2 macrophages scattered in the tongue muscle.The results showed that the number of M2macrophages in the TLR2-/-group mice was higher than the WT group.And with the progression of the tumor,the number of M2 macrophages infiltrating into the tongue rose in both groups.In vitro,we found that monocytes from TLR2-/-mice were more easily induced to M2 macrophages than WT mice.Conclusion1.During the entire process of the experiment?the process of tongue cancer?,TLR2-/-mice were always earlier than the WT group,and TLR2 knockout promoted the development of tongue tumorigenesis.2.The results showed that the number of M2 macrophages in the TLR2-/-group mice was higher than the WT group.In vitro,TLR2-/-macrophages are more easily induced to M2 macrophages than WT mice,that means TLR2 regulates the development of tongue carcinomas by regulating the differentiation and infiltration of M2 macrophages.