Compared with FN600, DN600 decreased direct GHGs (N2O + CH4) emissions by 21%, and increased the net GHG balance, GHG intensity, irrigation water-use efficiency (IWUE), and soil organic C content (ΔSOC) by 13%, 12%, 88%, and 89.8%, respectively. Higher costs in DN600  led to a 33.8% lower net profit than in FN600. Compared with FN600, DN420 reduced N and irrigation water by 30% and 46%, respectively, which increased partial factor productivity and IWUE , but DN420 did not affect GHG mitigation or net profit. Because lower profit is the key factor limiting the technical extension of fertigation, financial subsidies should be made available for farmers to install fertigation technology.In the wheat–maize rotation cultivation system in northern China, excessive irrigation and over-fertilization have depleted groundwater and increased nitrogen (N) losses. These problems can be addressed by optimized N fertilization and water-saving irrigation. We evaluated the effects of these practices on greenhouse gas emissions (GHG), net profit, and soil carbon (C) sequestration. We conducted a field experiment with flood irrigation (FN0, 0 kg N ha Many countries including China will have to adopt appropriate intensive production practices to ensure the sustainability of agriculture2. Globally, agricultural soil is thought to be one of the major contributors to greenhouse gas (GHG) emissions, accounting for 10%–12% of total anthropogenic GHGs3. However, agricultural soil also sequesters 4 × 108 − 9 × 108 Mg C yr−1 4.Agricultural sustainability implies that high crop yields can be maintained under extreme natural conditions and that agricultural practices have acceptable environmental impactsTriticum aestivum L.) and maize (Zea mays L.), respectively5. Currently, nitrogen (N) use by crops is still inefficient in this region, resulting in losses of up to 70% of applied fertilizer N6. If the agricultural N application rate (550–600 kg N ha) was reduced by 30%–60%, N losses may be decreased by more than 50%7. In Huantai County, northern China, agriculture is strongly dependent on pumped deep groundwater for irrigation, and the irrigation water-use efficiency (IWUE) is very low (<2.5 m−3 kg−1)8. The high annual depletion rate of groundwater (2.2 ± 0.3 cm) in this area is mainly due to the long-term overuse of water resources9. Since the 1990s, various sound farming options, e.g., (drip) fertigation, have been proposed and implemented to reduce the use of fertilizer N and irrigation water without decreasing the high crop yields in this region10.Northern China, one of the most intensified agricultural regions, accounts for only 25% of the farmland in China, but produces about 66% and 28% of the country’s wheat (11. Drip fertigation has been proven to decrease methane (CH4) emissions and N losses  and increase the soil organic carbon (SOC) content12, while maintaining similar crop yields to those achieved under flood irrigation14. However, some studies have also found that fertigation with similar N levels to those of flood irrigation may increase GHGs emissions11. This is because denitrification is stimulated by the increase in water-filled pore spaces (WFPS) due to frequent irrigation11. Besides the effects on direct GHGs , (drip) fertigation may also increase CO2 emissions because of increased energy consumption to run the system15 and to install the drip fertigation facilities16. In addition, large amounts of CO2 are produced during the manufacturing and transport of chemical fertilizers for crop production16. Compared with conventional flood irrigation, drip fertigation has different effects on the soil carbon (C) stock, because of differences in soil moisture content, temperature, and the initial decomposition rate of straw17. It is, therefore, critically important to comprehensively monitor and evaluate GHGs emissions, resource utilization, and land productivity of fertigation vs. flood irrigation, to optimize wheat/maize production in the intensified cropping region of northern China.Fertigation, where mineral fertilizer is supplied via a drip system, provides more appropriate amounts of nutrients  and water to the active plant root zone than does broadcast fertilizationIn view of the above, we established a field experiment in winter wheat–summer maize cropping system in northern China during 2015–2017, and monitored direct and indirect GHGs emissions, soil C sequestration, and production costs. The objectives of this study were to: (i) monitor the direct and indirect GHGs emissions and SOC stock changes with different irrigation and N fertilization combinations; (ii) assess the efficacy of optimized N fertilization and irrigation practices to mitigate GHGs emissions and maintain a high net profit; and (iii) provide suggestions and discuss the implications for sustainable agriculture in the future. On the basis of our results, we can propose strategies to improve the sustainability of agriculture in this region.Annual precipitation was higher in the 2015–2016 cropping year than in the 2016–2017 cropping year Fig. . CorrespBecause watering was more frequent in the drip fertigation treatments, the soil WFPS (0–10 cm depth) was higher in DN600 (65.2% ± 0.5%) than in the flood irrigation treatments (62.4% ± 1.3%) during the whole experiment. Consequently, soil temperatures (0–5 cm depth) were lower in the drip fertigation treatments  than in the flood irrigation treatments . The average WFPS of DN420, DN600, and FN600 was 64.9% ± 1.3%, 64.9% ± 1.3%, and 73.8% ± 1.1%, respectively, in the 2015–2016 cropping year, and 67.5% ± 1.2%, 68.9% ± 1.1%, and 80.2% ± 2.3%, respectively, in the 2016–2017 year, within 7 days after each irrigation event Table .Compared with the local conventional flood irrigation and fertilization practice, i.e., the FN600 treatment, DN600 resulted in higher yields of winter wheat and summer maize in both the 2015–2016 and 2016–2017 growing seasons , and significantly increased IWUE by 94% (P < 0.05) and PFP by 49% (P < 0.05), compared with their respective values in FN600  and 81.7% (flood) of the observations were negative fluxes, i.e., CH4 uptake or consumption. The maize season accounted for 40%–44% (2015–2016) and 36%–45% (2016–2017) of total annual CH4 uptake. Compared with the FN600 treatment, the DN600 treatment increased CH4 uptake by 31.8% (P < 0.05) in 2015–2016, and by 41.3% (P < 0.05) in 2016–2017  and by 5.2% in 2016–2017.The CHnts Fig. . Of all 017 Fig. , a mean 017 Fig. . Compare2O flux peaked within about 3 days after fertilizer application in the wheat growing season, but within 2 days after fertilizer application in the maize growing season. Fewer N2O flux events occurred from December to March, corresponding to the low soil temperature (−4.9–9.4 °C). The details of N2O fluxes in 2015–2016 have been described in our previous study. The maize season accounted for 63%–75% (2015–2016) and 63%–70% (2016–2017) of total annual N2O emissions , 2.67 , 1.7 , and 3.16 kg N2O-N ha−1 . Compared with the FN600 treatment, the DN600 treatment decreased the wheat seasonal N2O emissions by 34.7% in 2016–2017 (P < 0.05) and by 16.1% in 2015–2016; and the DN600 treatment decreased the maize seasonal N2O emissions by 20.2% in 2016 (P < 0.05) and by 17.1% in 2017 (P < 0.05). Compared with the DN600 treatment, the DN420 treatment decreased the wheat seasonal N2O emissions by 21.8% (2015–2016) and 17.1% (2016–2017), and decreased the maize seasonal N2O emissions by 31.5% (2016) and 28.6% (2017)  emissions in the FN600 treatment were 1.88 Mg CO2-eq ha−1 yr−1, which was 26.2% higher than in DN600 (P < 0.05) and 44.1% higher than in DN420 (P < 0.05) , mainly because of the higher electricity consumption and polyethylene required for irrigation facilities in DN600 (Table 2-eq ha−1 yr−1) than in FN600 and DN420, respectively. These results show that, in the life-cycle of wheat and maize production, indirect GHGs are main contributors to overall climate effects.The annual direct GHGs (N05) Fig. . In cont−1, respectively, significantly more than in FN600   and DN600 (0.86 Mg C ha−1 yr−1).After the 2-year experiment, the SOC content (0–30 cm) in DN420 and DN600 had increased by 0.52 and 0.41 g kg2-eq ha−1 yr−1) than in FN600. However, the total net GHG balance of DN420 was 15.8% and 4.9% lower than those of DN600 and FN600, respectively.The annual net GHG balance values were greater than zero for all management systems, indicating that all systems were net GHGs sources Table . Over th2-eq Mg−1 grain) were 12.1% and 1.3% higher, respectively, than those in FN600. The GHGI and yield in DN420 were 15.7% lower and 3.2% higher, respectively, than those in DN600.The net GHG balance per unit grain yield is usually defined as GHGI. The GHGI and yield in DN600  tend to stimulate methanotrophy25 and decrease N2O production27. At each drip fertigation event, the urea was directly applied to the crop rhizosphere with water at a greater frequency (five times for wheat and six times for maize) but at a lower rate (<70 kg N ha−1) than in the FN600 treatment . This may have caused the higher CH4 uptake in DN600 and DN420 than in FN600. As discussed in our previous study, within 7 days after fertilization, the soil inorganic N pool was significantly higher in flood-irrigated soils (91% higher in the wheat season and 55% higher in the maize season) than in drip-irrigated soils17. This increased the N-use efficiency, which would have decreased the level of soil inorganic N (NO3−-N + NH4+-N) available for transformation into N2O by microorganisms in the drip fertigation system13, as proven in previous studies28. As discussed in our previous study, in addition to nitrification oxidation, nitrifier denitrification also substantially contributes to the generation and diffusion of N2O, and after oxidation of NH4+ to NO2−, more N is transformed into N2O and NO than into NO3−17. In the present study, the soil NO3− content during the 2-year period was 24% lower in FN600 (40 mg kg−1) than in DN600 (53 mg kg−1), indicating that more soil NO2− was transformed into N2O or NO via nitrifier denitrification in FN600 than in DN600. This has been discussed in our previous paper17.In agricultural ecosystems such as cropland, fertilizer N may promote nitrification and denitrification by nitrifiers to produce N2O emission is 70%–80%14. The production of N2O via denitrification occurs at WFPS > 70%, whereas N2O is produced via nitrification at WFPS < 70%29. CH4 can be produced only in strictly anaerobic conditions30. A lower WFPS  has been shown to increase soil aeration, promote NH2OH oxidation, increase the conversion of N2O into NO31, and increase CH4 oxidation30. Additionally, N2O and CH4 emissions are known to be strongly and positively correlated with soil temperature4. Thus, the higher temperature in FN600 than in DN600 (19.2 °C vs. 17.8 °C) promoted N2O and CH4 production17.The optimum WFPS range for N15, thus contributing to greater CH4 uptake and reduced N2O emission32. As Lal33 reported, the rate of SOC accrual was only 0.3–0.5 Mg C ha−1 yr−1 under intensive agricultural practices, but was able to reach 1.0–1.5 Mg C ha−1 yr−1 in irrigated farming if sufficient straw was incorporated into soil. In this study, the rate of increase in SOC content was much higher in DN600 (0.45 g kg−1 yr−1) than in FN600 (0.20 g kg−1 yr−1). There are a number of factors that may have contributed to the high rate of SOC increase in DN600. Firstly, fertigation in DN600 may have contributed to greater root biomass, thereby strengthening the major pathway of C input into soil12. Secondly, a large amount of crop residues was returned to the soil in DN600. Crop residues are a key factor in determining changes in SOC stocks16. Here, more organic materials (straw) were returned to soil in DN600  than in FN600 . Thirdly, the initial SOC content in our study was lower than those reported for European and US cropland soils (14.5–23.2 g kg−1)16. Finally, the duration of our study (2 years) was shorter than those of other field studies (5–15 years)34.Optimum farming systems may result in greater soil C sequestration as SOC16. The ΔSOC and electricity consumption for irrigation were the main reasons for the differences in indirect GHGs emissions between drip and flood irrigation treatments  treatment significantly alleviated the higher direct + indirect GHGs emissions. Thus, DN420 was the best practice in terms of GHGs emissions and C sequestration, while DN600 showed the worst performance because the indirect GHGs emissions were higher than those in FN600. However, there was no significant benefit for GHG mitigation in DN420 because of the higher indirect GHGs emissions related to polythene and electricity consumption for irrigation. Therefore, some measures  to mitigate indirect GHGs emissions should be developed to promote the extension of fertigation in northern China.Analyses of the overall effects of agricultural practices on climate should include measurements of direct and indirect GHGs emissions and SOC changests Table , similar10 in northern China, because the economic output of vegetables (>$22000 ha−1) is much higher than that of wheat and maize . In Huantai County, the fertigation area was 1300 ha in 2017, with financial subsidies of up to 900 CNY per hectare. Our study highlights that other policies and financial schemes should be adopted to promote the environmentally friendly practice of optimized fertigation in China. For instance, in the 1990s, a pioneering financial subsidy policy in Huantai County helped farmers and cooperatives to purchase agricultural machinery for water-saving irrigation. The reduction in the amount of N fertilizer applied not only reduced the direct GHGs emissions from the soil, but also reduced the indirect GHGs emissions from production and transport, and reduced the input costs. For intensive agriculture in a semi-arid area, crops are very dependent on irrigation and chemical fertilizer . Meanwhile, saving irrigation water combined with optimized fertilizer use is proven to be the key to decreasing underground water usage and soil N losses, while increasing crop yields and water- and N-use efficienciesWe evaluated different systems combining drip or flood irrigation with synthetic N fertilizer for winter wheat and summer maize production in northern China. The different systems resulted in differences in crop productivity, N-use and water-use efficiencies, GHGs emissions, and SOC stock enhancement. Compared with the flood irrigation treatment, optimized drip fertigation (DN420) maintained a lower WFPS and soil temperature, and a higher soil SOC content throughout the whole experimental period. Compared with the local conventional fertilization and flood irrigation treatments, the DN420 treatment also resulted in higher crop yield, IWUE, and PFP. However, compared with the flood irrigation system, the fertigation system had no significant benefit in terms of GHGs mitigation, because of higher indirect GHGs emissions (associated with polyethylene and electric power). The higher inputs of electric power, polyethylene, and labour led to 26.4%–33.8% lower net profits in the fertigation system than in the FN600 treatment. Consequently, the extension of fertigation technology is limited in northern China. For future sustainable and environmentally friendly agriculture, optimized drip fertigation is highly recommended in the intensively farmed wheat and maize production regions of northern China. Policies and financial programs are required to support the installation of fertigation technology. The overall aim is to ensure that farmers can afford to implement an agricultural system that can respond to global climate change, and safeguard food security.17. The main cropping pattern in Huantai is the annual double cropping of winter wheat (T. aestivum L.) and summer maize (Z. mays L.). The region has a typical monsoon climate, with annual mean air temperature and precipitation of 12.5 °C and 542.8 mm, respectively. The experiment was conducted from Oct. 2015 to Oct. 2017  of 7.8, SOC content of 17.3 g kg−1, and total N content of 1.1 g kg−1. Since the 1980s, land productivity in this region has increased significantly with high rates of fertilizer application, frequent irrigation, and the cultivation of high-yielding varieties.The field experiment was conducted at Huantai Experimental Station, China Agricultural University, Shandong province −1 yr−1); (2) local farmers’ conventional level of N fertilizer and drip fertigation ; (3) optimal level of N fertilizer and drip fertigation ; (4) no N fertilizer and drip irrigation ; and (5) no N fertilizer and flood irrigation . Straw from the previous maize crop was mechanically chopped into pieces (5–8 cm length) and then incorporated into the field. Wheat (Luyuan 502) seeds were sown  in mid-October and mature plants were harvested in early June of the following year. The wheat straw was mechanically chopped (2–5 cm length) and then incorporated into the field. Maize (Zhengdan 958) seeds were sown  in mid- to late June and mature plants were harvested at the end of September.A completely randomized design was arranged with five treatments and three replicates, giving a total of 15 plots (each 10-m long and 5-m wide). The five treatments represented five different types of water and fertilizer N management, as follows: (1) local farmers’ conventional level of N fertilizer and flood irrigation  was applied as basal fertilizer at 118.3 kg K2O ha−1 for wheat and 84.7 kg K2O ha−1 for maize. Super phosphate (16% P2O5) was applied as basal fertilizer at 84.7 kg P2O5 ha−1 for wheat and 189 kg P2O5 ha−1 for maize. Table For fertigation treatments, fields were irrigated by a surface drip irrigation system, with one line in each row of wheat/maize. Pressure-compensating emitters were spaced 30 cm apart. In the winter wheat and summer maize seasons, N fertilizer  and irrigation events were as described in the Supplementary Materials. Potassium sulphate (52% K2O and CH4 fluxes were collected in situ simultaneously using a closed chamber method. The chamber was inserted into the soil (20 cm) under an emitter of one irrigation line in each experimental plot. During the sampling day, five air samples were taken at 8-min intervals while the chamber was closed using 35-ml polypropylene syringes and then stored in glass vials (30 ml). Gas was sampled from 8:00 to 11:00 local time in the morning for a continuous duration of 7–14 days following fertilization, rainfall, tillage and irrigation events, and twice per week during other periods. The N2O and CH4 levels in the samples were analysed with an Agilent 7820A gas chromatograph  within 24 h of sampling. The GHG fluxes were calculated from the five gas concentrations by nonlinear or linear methods, as described by Wang et al.36.From Oct 20, 2015 to Oct 10, 2017, N−1; kg P2O5 ha−1, kg K2O ha−1) rates were calculated according to the experiment design. Power use for irrigation  was calculated as follows:3 ha−1), and D is the water yield (m3 h−1). Fuel (kg ha−1) was the total diesel used for sowing and harvesting. Pesticides (kg ha−1), polyethylene lines in drip fertigation (kg ha−1), seeds , and labour (day person−1 ha−1) were recorded for each event.For indirect GHGs, chemical fertilizer  temperatures were measured with a digital thermometer .3− − N and NH4+ − N) content, topsoil (0–10 cm) samples were collected at the same time that air samples were collected. Topsoil samples were collected from two different locations: under the dripper and from mid-way between adjacent drippers. This sampling method accurately represents water distribution around the crop plant for soil moisture detection37. The proportion of WFPS (%) was calculated using Eq. (17:−3).To determine WFPS and soil inorganic N  after extraction with 1 M KCl (soil: solution = 1:5).The NH−1 HCl solution for 24 h to remove carbonates and oven-drying at 65 °C. Bulk density (g cm−3) was measured using the conventional core method. The annual topsoil (0–30 cm) SOC sequestration rate  was estimated from the change in topsoil SOC density  using Eq. (16:−3) of 0–30 cm depth topsoil; and 12 and 44 are the molecular weights of C and CO2, respectively.Here, we used soil organic carbon (SOC) content to represent C sequestered by soil. To determine the changes in SOC after the 2-year experimental period, composite soil samples were collected from each plot using a steel cylinder (5 cm diameter) at depths of 0–10, 10–20, and 20–30 cm. Soil samples were collected once (October) in 2015, twice (June and October) in 2016, and twice (June and October) in 2017, and air-dried and sieved through 0.5 mm mesh. The SOC content was determined with a CN analyser  after immersion of soil in 0.3 mol L−1) was defined as the ratio of crop yield to mineral fertilizer N applied10. The IWUE (kg m−3) was calculated as the ratio of grain yield  to the total irrigation water consumed , as follows17:Yg and Nfert are the grain yield (kg ha−1) and mineral fertilizer N rate (kg N ha−1), respectively.The partial factor productivity for N fertilizer  includes: (1) direct GHGs (soil N2O and CH4 emissions), and (2) indirect GHGs, i.e., upstream CO2 emissions from agricultural inputs , and (3) SOC stock changes (0–30 cm layer) during the experimental period30. Hence, the net GHG balance and GHG intensity (GHGI) were calculated as follows16:Direct (kg CO2-eq ha−1) = GWPN2O + GWPCH4, and represents the total CO2 emission equivalalents for soil N2O and CH4; GWPIndirect (kg CO2-eq ha−1) = GWPFertilizer + GWPElectricity + GWPFuel + GWPPesticide + GWPPE + GWPSeed + GWPLabor, and represents the total CO2 emission equivalalents for chemical fertilizer  inputs, power use for irrigation, fuel, pesticides, polyethylene lines in drip fertigation, seeds (of wheat and maize), and labour, respectively , and  SOC stoc2-eq Mg−1 grain yield) represents the net GHG balance for production of a unit weight (Mg) of grain15, and was calculated as follows:The GHGI (kg COP < 0.05) in one-way analysis of variance (ANOVA). Differences were considered significant at P < 0.05.All data were analysed using SPSS Statistics 22.0 software  and plotted with Microsoft Excel 2016. Statistically significant differences were detected using the least significant differences (LSD) method (Supplementary Information.


Spontaneous functional recovery occurs during the acute phase after stroke onset, but this intrinsic recovery remains limited. Therefore, exploring the mechanism underlying spontaneous recovery and identifying potential strategies to promote functional rehabilitation after stroke are very important. The CD200/CD200R signaling pathway plays an important role in neurological recovery by modulating synaptic plasticity during multiple brain disorders. However, the effect and mechanism of action of the CD200/CD200R pathway in spontaneous functional recovery after stroke are unclear.In this study, we used a transient middle cerebral artery occlusion (MCAO) model in rats to investigate the function of CD200/CD200R signaling in spontaneous functional recovery after stroke. We performed a battery of behavioral tests  to evaluate sensorimotor function after intracerebroventricular (i.c.v.) injection with CD200 fusion protein (CD200Fc) or CD200R blocking antibody (CD200R Ab) post-stroke. Density and morphology of dendritic spines were analyzed by Golgi staining. Microglia activation was evaluated by immunofluorescence staining. Western blot was used to detect the levels of protein and the levels of mRNA were measured by qPCR.Il-1, Tnf-α, and Il-6) after MCAO. On the contrary, CD200R Ab aggravated sensory function recovery in adhesive removal test and further promoted microglia activation and pro-inflammatory factor release (such as Il-1) after MCAO. The immune-modulatory effect of CD200/CD200R signaling might be exerted partly by its inhibition of the MAPK pathway.Our study demonstrated that sensorimotor function, synaptic proteins, and structures were gradually recovered and CD200R was transiently upregulated in ipsilateral cortex after stroke. Synapse-related proteins and dendritic spines were preserved, accompanied by sensorimotor functional recovery, after stereotaxic CD200Fc injection post-stroke. In addition, CD200Fc restrained microglia activation and pro-inflammatory factor release (such as This study provides evidence that the CD200/CD200R signaling pathway contributes to spontaneous functional recovery by enhancing synaptic plasticity via inhibition of microglia activation and inflammatory factor release. Stroke, among which ischemic stroke accounts for nearly 87%, is a leading cause of death and disability worldwide, causing ~ 80% of all disabilities . Due to The integrity of neuronal networks is the basis of sensorimotor behavior; however, neuronal networks and synapse circuits are damaged by stroke in regions that receive reduced blood supply, which results in sensorimotor dysfunction . The surIl-1, Tnf-α, and Il-6) can be released by microglia in response to synapse degeneration or loss [Cd200) impairs long-term potentiation (LTP) [Inflammation plays an important role in regulating synaptic plasticity –9. Activ or loss , 11. Thu or loss  and also or loss –15. The on (LTP) , whereason (LTP) –19. HoweHere, we propose that the CD200/CD200R signaling pathway contributes to spontaneous functional recovery after stroke by restraining synapse loss through inhibiting microglia activation and pro-inflammatory factor release.Adult male Sprague-Dawley (SD) rats  were purchased from Shanghai Sippr-BK laboratory animal Co. Ltd.  and housed in the Pharmaceutical animal experimental center of China Pharmaceutical University under a 12 light–dark cycle with food and water ad libitum. All animal procedures were approved by the Animal Research Ethics Committee of China Pharmaceutical University.A transient right middle cerebral artery occlusion (MCAO) stroke was performed as previously described . BrieflyThe animals were anesthetized with 2 % isoflurane and placed on a stereotaxic apparatus . After exposing the skull, a small hole was made to allow the intracerebroventricular injection. CD200Fc   as an agonist of CD200R1, anti-CD200R1 antibody (CD200R Ab)  as an antagonist of CD200R1 (5 μL), or IgG  was injected into the right cerebral ventricle one time by a 10-μL Hamilton syringe 24 h after MCAO. The injection was controlled at a rate of 0.5 μL/min and the syringe remained in place for 5 min before the completion of injection. The stereotaxic coordinates of the right cerebral ventricle were as follows: bregma − 0.8 mm; lateral − 1.5 mm; ventral − 4.0 mm.Behavioral tests were performed at 4, 7, 14, 21, and 28 days after MCAO by an investigator blinded to the experimental groups.This test was assessed using a 5-point scale as described previously : 0, no oTo measure the sensory functions and motor functions, the tests were performed as described previously  with somTo evaluate forelimb-use asymmetry, rats were placed in a transparent cylinder (diameter 20 cm and height 45 cm). The times of touching the wall by the forelimb of rats were recorded. The detailed operation was described previously .Rats were hung to a horizontal rope  by its forepaws to evaluate the muscle strength . Time ofRats were anesthetized with ketamine and xylazine in 0.9% saline at 4, 7, and 28 days after MCAO and perfused with 0.9% saline, followed by fresh cold 4% PFA in 0.9% saline. Brain tissues were fixed in fresh 4% PFA 4 °C overnight and dehydrated gradient in 20% and 30% sucrose at 4 °C before sectioning on a Leica-1950 cryostat  at 10 μm and 30 μm. Sections were stored at − 20 °C for immunohistochemistry.Cerebral infarct volume was detected at 4 days, 7 days, and 28 days after MCAO . Rats weBrain sections were permeabilized by 0.3% Triton-100 in PBS, blocked by 10% goat serum in 90% PBS at room temperature for 1 h, and then incubated primary antibody at 4 °C overnight. The primary antibodies were as follows: mouse anti-CD200R , mouse anti-CD68 , and anti-Iba1 . After rinsed three times, sections were incubated with Alexa Flour 488 conjugated goat anti-rabbit IgG  and Alexa Flour 633 conjugated goat anti-mouse IgG  at room temperature for 1 h. The fluorescent imaging was collected by an Olympus fluorescence microscope and processed by ImageJ software.Animals were sacrificed and brain tissues were removed at 28 days after MCAO. Samples were immersed in Golgi-Cox solutions for 2 weeks and then dehydrated gradient in 20% and 30% sucrose at 4 °C. Tissues were cut into 100-μm sections by Leica-1950 cryostat  for following staining. Sections were immersed in 75% ammonia solution and 1% sodium thiosulfate. Images were collected on a microscope, and 10~20 neurons per sample were randomly observed for morphological analysis.2 and 95% oxygen. BV2 were treated with IgG (15 μg/ml) or CD200Fc (15 μg/ml) 30 min before OGD. BV2 were subjected to OGD as previously reported [2, 5 ml HEPES, and 1.125 g glycine in a total volume of 500 ml. Then, BV2 were kept in a humidified atmosphere containing 95% nitrogen and 5% CO2 for 6 h and reoxygenation for 16 h. For reoxygenation, OGD buffer was replaced with BV2 culture medium and cells were returned to normoxic conditions (95% air and 5% CO2). BV2 cultured under the normoxic condition served as the control.Cells from the immortalized mouse microglia cell line BV-2 were cultured in DMEM containing 10% fetal bovine serum (FBS) and maintained at 37 °C in 5% COreported , 26 withTo determine whether CD200Fc could inhibit JNK and P38 phosphorylation in BV2 cell, CD200Fc (15 μg/ml) or IgG (15 μg/ml) in the presence of anisomycin (1 nM) was added to the BV2 for 30 min, and BV2 cultured under the normoxic condition served as the control.At 4, 7, and 28 days after MCAO, brain samples were collected from ipsilateral sensorimotor cortex. Total RNA was isolated from frozen tissues using TRIzol Reagent  according to the manufacturer’s protocol. Equal amounts of total RNA were reverse transcribed under standard condition using HiScript 1st Strand cDNA Synthesis Kit . Quantitative PCR was performed on an ABI7000 real-time PCR system  using SYBR Green Master Mix . The cycle time values were normalized to GAPDH of the same sample. Primer sequences are shown in Table At 4, 7, and 28 days after MCAO, brain samples were collected from the ipsilateral sensorimotor cortex. Brain tissues were homogenized with RIPA lysate  supplied with protease inhibitor cocktail . An equal amount of protein was separated on SDS polyacrylamide gels, transferred to polyvinyl-difluoride membranes , and blocked with 2.5% (w/v) bovine serum albumin (BSA). The membrane was incubated with primary antibody  at 4 °C overnight. After being rinsed three times (7 min/time) with TBST, the membrane was incubated corresponding secondary antibody at room temperature for 1 h. After washed, the labeled proteins were detected using the Bio-Rad system .P < 0.05.The GraphPad Prism Software 6  was used for statistical analysis, and the results were presented as a mean ± SEM. Among the frequencies, Longa test Fig. a was anaP < 0.05)  and spent more time removing the spot at 4, 7, 14, and 21 days after MCAO compared with the sham group      Fig. a. In the32) Fig. . The tim67) Fig. . The hanF3, 24 = 101.0, P < 0.0001)  and SYP  were decreased significantly at 4, 7, and 28 days after MCAO compared with the sham group  , the percent stubby spine was increased significantly, and the percent mushroom spine was decreased significantly at 28 days after MCAO     Fig. . These rP < 0.05)       injection with CD200 fusion protein (CD200Fc) or CD200R blocking antibody (CD200R Ab) post-stroke. First, CD200Fc significantly improved neurological recovery in Longa tests at 4 and 7 days after MCAO, whereas neurological recovery was restrained at 4 days after CD200R Ab injection compared with the MCAO+IgG group (01) Fig. . We alsoF4, 30 = 101.1, P < 0.0001), and SYP levels were increased at 28 days  after CD200Fc i.c.v injection post-stroke  , and spine shape was altered from stubby to mushroom by CD200Fc application at 28 days after MCAO   release. The number of Iba1+/CD68+ cells in the ipsilateral sensorimotor cortex was decreased markedly at 4 and 7 days after MCAO in the MCAO+CD200Fc group compared with the MCAO+IgG group    (P < 0.05)   injury and neurodegenerative diseases , 31, andκB and mitogen-activated protein kinase (MAPK) pathways [F4, 30 = 26.20, P < 0.0001)   Fig. a, b, butF2, 6 = 0.009141, P = 0.9909; p-P38, F2, 6 = 1.289, P = 0.3423). Next, we further determined the effect of CD200Fc on JNK and P38 phosphorylation in microglia via anisomycin (agonists of these JNK and P38), and the results showed that CD200Fc significantly decreased the protein level of p-JNK and p-p38 induced by anisomycin . These data indicated that CD200Fc could inhibit JNK and p38 phosphorylation in microglia.To test whether CD200Fc inhibited microglial activation is mediated via MAPKs especially JNK and p38, we used BV2 cells to test the effect of CD200Fc on JNK and P38 phosphorylation after OGD. As shown in Fig. Recent studies indicated that the CD200/CD200R signaling pathway regulates the microglia activation-induced inflammatory response and synaptic plasticity in vitro and in vivo , 35, 37,Ischemic stroke impairs the integrity of neural networks and intercellular signal transmission and causes sensorimotor deficits . SurviviThe CD200/CD200R signaling pathway regulates synaptic plasticity, and dysfunction in this pathway contributes to synaptic deficits in aging and AD , 29. In Il-1β, Il-6, and Tnf-α) factors and increasing the mRNA levels of anti-inflammatory (Il-4) regulators in the ipsilateral cortex. Similar effects were also observed after CD200Fc application in LPS-stimulated microglia cells, during aging, and in Parkinson’s disease [Inflammation and pro-inflammatory factors are secondary injury mediators following cerebral ischemia , and the disease , 45. TheκB is an important transcription factor that promotes microglia activation and inflammatory factor release [κB pathway in vitro [κB pathway after stroke. CD200R is only expressed in microglia in the CNS, whereas inflammation is a complex response involving many kinds of cells. Activating CD200/CD200R signaling may be insufficient to contain the NF-κB pathway after stroke in vivo. Recent studies reported that the CD200/CD200R pathway modulated the inflammatory response by controlling MAPK activation in vitro and in vivo [NF- release , 47. Prein vitro . However in vivo –51. The One limitation of the present study is that CD200R not only expressed in microglia, but also expressed in peripheral myeloid cells  and neurThe CD200/CD200R signaling pathway participates in spontaneous functional recovery after stroke. Activation of CD200/CD200R signaling attenuated brain injury and facilitated the recovery of sensorimotor function. These effects were exerted by increasing synaptic plasticity via inhibiting microglia activation and improving the inflammatory microenvironment Fig. . Therefo


THRA. The entity was first described in 2012 and to date only a small number of patients with varying severity have been reported. In this review, we summarize and interpret the heterogeneous clinical and laboratory features of all published cases, including ours. Many symptoms and findings are similar to those seen in primary hypothyroidism. However, thyroid-stimulating hormone levels are normal. Free triiodothyronine (T3) levels are in the upper half of normal range or frankly high and free thyroxine (T4) levels are low or in the lower half of normal range. Alterations in free T3 and free T4 may not be remarkable, particularly in adults, possibly contributing to underdiagnosis. In such patients, low reverse T3 levels, normo- or macrocytic anemia or, particularly in children, mildly elevated creatine kinase levels would warrant THRA sequencing. Treatment with L-thyroxine results in improvement of some clinical findings.Resistance to thyroid hormone alpha occurs due to pathogenic, heterozygous variants in  The thyroid gland has important roles in energy homeostasis, skeletal growth, cardiac and gastrointestinal function, and maturation of the central nervous system . ThyrotrTHRA (chromosome 17) and THRB (chromosome 3), respectively. TRα has two isoforms produced with alternative splicing. TRα1 is mainly expressed in the central nervous system, bone, myocardium, skeletal muscle and gastrointestinal tract, while TRα2 is expressed in various tissues but has no binding site for T3 and thus its function is enigmatic  in the central nervous system . Intracenigmatic ,7. TRβ1 nigmatic ,7,8.THRB took more than two decades . The incidence of RTHβ is reported to be approximately 1/40000 and is characterized by goiter, tachycardia, hyperactivity, failure to thrive and cognitive impairments with high serum TH levels, but normal or mildly elevated TSH  . The fir decades . Pathogeated TSH ,13,14. Tva et al . To dateublished , 2 . TRα and TRβ act via transcriptional repressors, such as nuclear receptor corepressor-1 (NCoR1), in the absence of T3. This effect results in modification of histone deacetylase (HDAC) enzymes into a co-repressor complex, which suppresses basal T3 target gene transcription with remodeling of chromatin . When T3If TRα is mutant, it cannot release NCoR1 as a response to T3. Consequently, T3 target gene transcription remains suppressed because of the inhibition of wild-type TR through constant HDAC-induced chromatin remodeling. In the light of this molecular information, RTHα demonstrates clinical features with reduced T3 action in related tissues. In addition, a dominant-negative potential of the mutant TRα determines the severity of disease .The first experimental study of TRα was reported in 1997, 15 years before the first human cases were reported, showing that a TRα knock-out mouse had postnatal growth arrest with delayed maturation in small intestine and bones .THRA variants during genetic analyses for autism spectrum disorder . Ten of the 12 short children did not receive L-thyroxine (LT4) therapy before diagnosis and the lowest height standard deviation (SD) score was -3.1 ,25,28,29th ribs and scoliosis). However, these findings were atypical for RTHα and have not been reported in any other case to date. Bone mineral density was reported to be normal in three adult patients (Wormian bones in skull sutures were present in 10 among 31 cases with available data (32%) ,24,25. Vpatients .Normally, tooth eruption is expected to occur before 13 months of age . DelayedBochukova et al  reportedT3 and its receptors play a major role in neuronal migration, synaptogenesis, maturation, myelination and differentiation of oligodendrocytes or glial cells . That isIn infants with RTHα, delayed milestones for motor and speech abilities are the most common symptoms, noted in 34 among 40 cases (85%) 30,31,32.Furthermore, Demir et al  reportedSeizures after stimulation with light or audio and abnormal evolution of GABAergic neurons in TRα1 mutant mice correlated with epilepsy in human cases . To date, three cases with RTHα were reported to be suffering from epileptic seizures in childhood ,23,32.THRA variants in six of them. Unfortunately, no additional clinical data regarding these children were provided. Coexistence of autism spectrum disorder and RTHα was reported in two more patients, suggesting that RTHα should be excluded in patients with autism spectrum disorder  separately from the patients who were analyzed after discontinuation of LT4 treatment.A normal neonatal congenital hypothyroidism screening result  was reported in a case with RTHα, who also had an uneventful neonatal period .TSH levels were all normal in affected children. Among the adult patients, an atypical case with severe malformations was the only one with abnormal TSH   22)..22).Differences of TH levels among treatment-naive children and adults are also shown in Both fT4 and TSH were normal in 61% (11 among 18) of children and 78% (7 among 9) of adults. Normal fT3, fT4 and TSH were noted in 33% (5 among 15) and 83% (5 among 6) of children and adults, respectively . In suchAfter cessation of LT4 treatment, mildly elevated TSH may be seen, as was reported in one adult and one child with RTHα ,18. The Under LT4 treatment, fT3 and fT4 levels increased in patients with RTHα, while TSH was suppressed, a similar pattern to that found during the treatment of central hypothyroidism ,23,24,29The relationship between anemia and hypothyroidism is well-known (68). Animal models lacking TRα demonstrated compromised erythropoiesis ,70. In aAn increase in serum levels of interleukin-8 (IL-8), a pro-inflammatory cytokine, was shown in RTHα patients. However, neutrophil or macrophage functions, which are partly mediated by IL-8, were found to be normal in those cases .Both thyroglobulin and urinary iodine levels are expected to be in the normal range 34). Similar to primary hypothyroidism, high total cholesterol and LDL levels, and low or low-normal levels of IGF-1 can be found in RTHα ,34.. SimilarIn primary hypothyroidism, creatinine kinase (CK) can also be elevated . Human dRecently, Boumaza et al 74) reported that biofluids (urine and plasma samples) of TRα-mutant mice showed distinct metabolomic profiles from controls, including increased urinary levels of hippurate and decreased urinary levels of isovalerylglycine, dimethylamine, trimethylamine, and choline. They suggested that easily accessible nuclear magnetic resonance-based metabolic fingerprints of biofluids could be used to diagnose RTHα in humans . However, clinical and laboratory signs of peripheral thyrotoxicosis are present in this disease in addition to cerebral hypothyroidism ,77,78,79or mildly elevated CK or low rT3. Among the subjects with available data, the algorithm in or mildly elevated CK values or low rT3 levels would be an additional clue leading to THRA sequencing. This approach is valid for 10 of 12 adult patients with available data (83%)  ,23,24,30There is only limited data about the treatment of RTHα and thus long-term follow-up data is required. LT4 treatment has been the first choice to date, in order to overcome the resistance in TRα with higher dosage. T4 and rT3 levels come into the normal range with this treatment and T3 level remains high. Since the feedback mechanism of the HPT axis is intact, LT4 treatment causes TSH suppression in RTHα patients ,23,24,29In animal models with mutant TRα, increasing serum TH levels alleviated locomotor and behavioral irregularities . TherefoVan Mullem et al  reportedWith the peripheral effects of LT4 treatment, increases in sex hormone binding globulin (SHBG) or IGF-1 levels can be seen, as previously reported in RTHα patients. In addition to this, CK or cholesterol levels were reduced in these cases, reflecting the improved tissue response to TH ,23,24,25Anemia seems to be unresponsive to LT4 treatment, as described in most of the RTHα cases ,19,25,29THRA, including the carboxy-terminal part of TRα1, had varying responses to LT4 treatment. Like their severity of clinical presentation, this situation was also associated with the specific location of the variant or the degree to which this molecular region was affected (Patients who had frameshift variants in affected ,18,24. Iaffected ,18,24.Since LT4 administration to RTHα patients will excessively stimulate TRβ in TRβ-dominant tissues, development of TRα1-selective thyromimetics would be ideal ,81. AlteThe diagnosis of RTHα is not straightforward since TH levels might not be helpful and the entity is not widely known. As published data is limited concerning RTHα, absence of phenotypic features or laboratory findings would not exclude RTHα. Currently, only fT4 and TSH levels are recommended for evaluation of growth failure in children . However


Pasteurella multocida is an important pathogenic bacterium of domestic animals. However, the mechanisms of infection are still poorly understood. Here, we found that Pm0442 was dramatically up-regulated in infected mice among 67 predicted lipoproteins of P. multocida serotype A CQ2 strain (PmCQ2). To explore the role of Pm0442 in virulence and the potential of the mutant as a vaccine, Pm0442 mutant of PmCQ2 was successfully constructed. Then, the virulence characteristics, immune/inflammatory responses, and the survival rates of challenged mice were determined. As a result, it was found that the Pm0442 deletion of PmCQ2 significantly decreased bacterial loads and inflammatory responses of lung tissue in mice, resulting in improved survival. Mechanically, Pm0442 affects PmCQ2 capsular and lipopolysaccharide (LPS) synthesis and iron utilization-related genes expression affecting adhesion and phagocytosis. Furthermore, PM0442 bound directly to Toll-like receptor 2 (TLR2) to mediate the secretion of pro-inflammatory cytokine  in macrophages via activation of the NF-κB, ERK1/2 and p38 signaling pathways. Notably, PmCQ2Δ0442 could provide 70–80% protection to mice challenged with 3.08 × 107 CFU of PmCQ2. Our findings demonstrate that Pm0442 is a virulence-related gene of PmCQ2, which provides new guidance for the prevention and control of Pasteurellosis. Pasteurella multocida (P. multocida) is a Gram-negative bacterium that causes diseases in poultry, livestock, and humans, resulting in huge economic losses  are capable of activating immune cells by recognizing the pathogen-associated molecular patterns (PAMPs) of bacteria, resulting in the expression and release of a variety of cytokines . LipoproPm0442 deletion of PmCQ2 significantly improved mouse survival, decreased bacterial loads and inflammatory responses of mice lungs. Mechanically, Pm0442 affects the expression of capsular, LPS, iron, and TonB receptor-related virulence genes. Furthermore, PM0442 induces the secretion of cytokines in macrophages though TLR2-NF-κB and MAPK signaling pathways. Additionally, PmCQ2Δ0442 provided 70–80% protection to mice challenged with PmCQ2 (3.08 × 107 CFU). These results provide a new insight of infection mechanism to P. multocida.In this study, we demonstrated that P. multocida capsular type A CQ2 (PmCQ2) is isolated from a lung of calf with pneumonia in Chongqing, China . Total 136 C57BL/6 WT, 32 Tlr2–/–, 24 Tlr4–/–, and 140 KM mice were used in this study. The mice were housed in individually ventilated pathogen-free cages  with free access to food and water. The mice were acclimated for 4 days after arrival before starting the experiments. This study was carried out in accordance with the principles of the Basel Declaration and recommendations of the Laboratory Animal Welfare and Ethical Commission of the Southwest University (Permit No. 11-1025), Chongqing, China.C57BL/6 and KM female mice (7–8 weeks old) were purchased from the Laboratory Animal Center of Third Military Medical University . C57BL/6 Tlr2CP033599.1) and transcriptome sequence (SRA accession: PRJNA597403) of P. multocida CQ2 strain, lipoproteins  resistance gene expression cassettes combined with a fragment containing origin of DNA replication from temperature-sensitive (TS) plasmid pCP109 . The cells were recovered at 30°C for 2 h and then spread onto Martin agar plates containing Kan (100 μg/mL) incubating for 24 h at 30°C. Individual colonies were transferred to 5 mL Martin broth with Kan (100 μg/mL) and incubated at 30°C for 24 h. 10 μL of broth from the transformed cell cultures was spread onto Martin agar plates with Kan (100 μg/mL) and grown at 42°C for 16 h to generate single-crossover mutants. Representative colonies were passed into 5 mL Martin broth lacking antibiotic and grown at 30°C for 16 h to allow resolution. The PmCQ2Δ0442 mutant was selected on Martin agar plates containing Kan (100 μg/mL) at 30°C. Subsequently, the candidate mutant clones were verified by PCR screening using primers Pm0442-F/Pm0442-R and pUC19-F/pUC19-R, as depicted in KpnI and EcoRI of plasmid pUC19 to generate the plasmid pUC19oriKanR. Next, the Pm0442 expression cassettes composed of a 162 bp upstream segment, a 45 bp downstream segment, and 690 bp Pm0442 were amplified from PmCQ2 genomic DNA with the primers pPm0442U-F and pPm0442D-R. Then the amplified fragment was inserted into the HindIII and BamHI sites of pUC19oriKanR to generate the complementary plasmid pUC19oriKanR-Pm0442. Then the plasmids were transformed into the PmCQ2Δ0442 mutant strain, generating the corresponding complementary strain PmCQ2Δ0442/pPm0442. Primers sequences are listed in To investigate whether d pCP109  were ins8 CFU in 20 μL, and the numbers of mice used for each strain detection were equal. Mice were monitored for 7 days to determine the survival curves, and mice showing severe clinical signs  were considered moribund and were humanely killed. Survival rates (n = 12/group) were measured in three groups after injection. Mice were also euthanized for collection of lung tissues to measure the bacterial loads (n = 10/group), RT-PCR (n = 6/group), and ELISA (n = 8/group), and serum samples (n = 8/group) at 16 h post-infection for ELISA  were randomly divided into 10 groups (n = 10/group). PmCQ2 experimental groups (5 groups) of mice were infected intramuscularly with 100 μL of various doses PmCQ2 . Experimental groups of PmCQ2Δ0442 (5 groups) were infected intramuscularly with 100 μL of various doses of PmCQ2Δ0442, ranging from 2.0 × 105 to 2.0 × 109 CFU. For all groups, mice were monitored for 7 days to determine the survival curves, and mice showing severe clinical signs were considered moribund and were humanely euthanized. Then the numbers of surviving mice in each group were recorded, and the LD50 was calculated with the method of Bliss (To determine the impact of of Bliss .n = 6/group) were immediately fixed in the 4% paraformaldehyde for 24 h, dehydrated in graded ethanol, and then embedded in paraffin wax. The tissues were sliced at 3 μm thick and then stained with hematoxylin and eosin (H&E). Histopathological scoring was performed by a pathologist blinded to treatment and control groups, and the scoring was mainly based on interstitial inflammation, vascular endothelialitis, bronchitis, edema, serous effusion and thrombus formation, all parameters were scored separately from 0 (lesion absent) to 3 (severe lesion)  .n = 10/group). Three experimental groups of mice were intramuscularly inoculated with 100 μL of different concentrations of PmCQ2Δ0442 , and the control group mice were inoculated with 100 μL PBS. The mice were intramuscularly infected with 3.08 × 107 CFU PmCQ2 at day-21 after inoculation. The mice were monitored for 10 days after challenge, which showing severe clinical signs were considered moribund and were humanely euthanized, and the numbers of surviving mice in each group were recorded.To determine immune protection of PmCQ2Δ0442, 40 female KM mice were randomly divided into four groups (n = 3) and PmCQ2Δ0442 (n = 3) at the dose of 1 × 108 CFU in 20 μL. Mice were euthanized at 16 h post-infection, lung tissues were collected and quickly frozen in liquid nitrogen. Simultaneously, one milliliter fresh mid-logarithmic phase of PmCQ2 (n = 3) and PmCQ2Δ0442 (n = 3) were seeded in 100 mL fresh Martin liquid medium and incubated at 37°C with shaking at 220 r/min for 12 h. Bacterial cultures were centrifuged at 3,000 r/min for 10 min at 4°C, the cell pellets were suspended in 10 mL ice-cold PBS and centrifuged again. The bacterial cells were quickly frozen in liquid nitrogen after washed three times. Lung tissues and bacteria samples were sent to the Beijing Genomics Institute  for transcriptome sequencing and analysis . The data in this study have been deposited to NCBI’s Sequence Read Archive (SRA) database and the accession number is PRJNA597831.To explore the details of PmCQ2Δ0442 virulence decrease, C57BL/6 mice were infected by an intranasal inoculation with PmCQ2 . Then the LPS content in bacterial culture and bacterial lysate were determined using an ELISA Kit , which is used for the quantification of LPS.–/–, and Tlr4–/– mice were injected intraperitoneally (i.p.) with 2mL 4% thioglycolate broth , and peritoneal macrophages were collected 3 days later. The peritoneal macrophages were suspended in RPMI 1640 medium  containing 10% FBS  and counted with a hemocytometer. The cells were seeded in 24-well microplates at a density of 5 × 105 cells/well and incubated at 37°C in 5% CO2. The non-adherent cells were removed after 2 h. Then adherent cells were challenged with PmCQ2, PmCQ2Δ0442 and PmCQ2Δ0442/pPm0442 at a multiplicity of infection (MOI) of 1 for 24 h. The supernatants were collected for cytokines detection by ELISA.Peritoneal macrophages were obtained according to previous methods . Briefly5 cells/well) with RPMI 1640 medium and challenged with P. multocida at an MOI of 1 for 3 h. Cells were washed three times with 4°C chilled PBS to remove non-associated bacteria and lysed in PBS containing 0.1% Triton X-100. Then the cell lysates were diluted with PBS and grown on Martin’s agar plates at 37°C for 18–24 h to determine the number of P. multocida associated with macrophage .According to the previously described method , brieflypm0442 gene was amplified and inserted into pET28a plasmids and induced for expression in Escherichia coli BL21(DE3). Purified recombinant PM0442 (rPM0442) proteins were prepared in accordance with previously described protocols . Then endotoxin was removed by using Toxin EraserTM endotoxin removal kit  and tested through ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit .To explore the function of PM0442 protein, the rotocols , and des5 cells/well), respectively, and the blank control (UI) macrophages received medium only. Then the peritoneal macrophages were cultured at 37°C, 5% CO2 for 24 h, and the supernatants and pellets were collected after centrifugation at 5,000 r/min for 5 min, the supernatants were used for the detection of secreted cytokines , and cell pellets were used for RNA extraction to detect the expression of TLRs by qRT-PCR.To investigate whether PM0442 protein is involved in the secretion of inflammatory cytokines from macrophages. Endotoxin-free rPM0442 proteins  and the parallel controlling factors including LPS , Boiled rPM0442  and Proteinase K (PK) + rPM0442 (50 μg/mL + 15 μg/mL) were added to the cell culture plate wells containing peritoneal macrophages  involving an gDNA elimination step. cDNAs were synthesized with an iScript cDNA synthesis kit , and quantitative real-time RT-PCR was performed according to previous study using a CFX96 instrument  . qRT-PCR6 cells/well in RPMI 1640 containing 10% FBS at 37°C for 2 h. After washing with PBS three times, the culture medium was replaced by RPMI 1640 without FBS, and the adherent cells were incubated with 10 μg rPM0442 protein. The cells were collected after 0, 5, 15, 30, and 60 min of incubation, and were lysed with radio-immunoprecipitation assay (RIPA) buffer . The cell lysates were subjected to SDS-PAGE and subsequently transferred to 0.2 μm polyvinylidene difluoride (PVDF) membranes by electroblotting. The membranes were immunoblotted with antibodies specific to IκBα, p-IκBα, NF-κB, p38, p-p38, ERK (1/2), p-ERK (1/2), JNK, p-JNK  and β-actin .Peritoneal macrophages were cultured in 12-well plates at a density of 1 × 10To further verify that rPM0442 protein induces the secretion of inflammatory cytokines in macrophages through MAPK and NF-κB signaling pathways. 10 μg endotoxin-free rPM0442 protein in RPMI 1640 medium containing 10% FBS was incubated with peritoneal macrophages washed three times with PBS. The culture supernatants were collected and used after 24 h of incubation. Inhibitors of NF-κB (BAY11-7082), JNK (SP600125), p38MAPK (SB203580), and ERK (U0126) were purchased from Beyotime , TLR2Ab from Thermo (United States), TLR4Ab from Santa (United States). All inhibitors or specific antibody were added to cultures 1 h before rPM0442 protein incubation. Levels of secreted cytokines in the culture supernatants were determined by ELISA. ELISA kits for IL-1β, TNF-α, IL-12p40, IL-6 were purchased from eBioscience .To investigate whether PM0442 protein directly binds to TLR2, the purified His-rPM0442 (500 μg) proteins were mixed with 100 μL of Ni-NTA (His-tag affinity) magnetic beads and gently mixed, parallel control was only containing 100 μL of Ni-NTA magnetic beads. The mixture was placed on a rotator and slowly mixed for 30 min, carefully removed the liquid on the magnetic stand. Next, the mixture was mixed with 500 μL peritoneal macrophage lysate and incubated overnight at 4°C on a rotator. Then the liquid was removed and washed three times with 4°C precooled PBS on a magnetic stand. Finally, it was re-suspended in 100 μL precooled PBS, which was the final sample of pull-down. The samples were subjected to SDS-PAGE and then detected using anti-His  and anti-TLR2 antibodies  with macrophage lysate and rPM0442 as control.t-test with Welch’s correction (Prism 6.0) if the data are in Gaussian distribution but show unequal variance, or by non-parametric test  if the data were not normally distributed. The Gaussian distribution of data was analyzed by D’Agootino-Pearson omnibus normality test (Prism 6.0) and Kolmogorov–Smirnov test (Prism 6.0). The variance of data was analyzed by Brown–Forsythe test (Prism 6.0). Differences with p < 0.05 were considered significant.Data were shown as the means ± standard deviation (SD). Data between two groups were analyzed using unpaired t tests (Prism 6.0) if the data were in Gaussian distribution and had equal variance, or by unpaired Pm0442 was highly expressed in mice infected by PmCQ2  was present in parent strain PmCQ2 and complemented strain PmCQ2Δ0442/pP0442, but not in the mutant strain PmCQ2Δ0442, and the positive control KMT1 could be amplified from three strains , respectively. Pm0442 was transcribed and expressed in the parent strain PmCQ2 and the complementary strain PmCQ2Δ0442/pP0442, but not in the mutant strain PmCQ2Δ0442 .Our previous findings indicated that by PmCQ2 , thus, wbination  and veri strains . TranscrCQ2Δ0442 , demonstPm0442 gene may affect the formation of PmCQ2 capsule.The parent strain PmCQ2 showed a typical growth curve, with a short lag phase (0–2 h), followed by a log phase during which major bacterial growth occurred (2–10 h) and then a stationary phase (10–14 h). In contrast, PmCQ2Δ0442 grew more slowly at 0–4 h, but the growth rate was reversed at 4–10 h. The OD values of PmCQ2Δ0442 were significantly lower than those of the parent strains between 0 and 8 h. The complementary strain PmCQ2Δ0442/pP0442 partially restored the defective growth, but the OD values remained lower than those of the parent strain . The col8 CFU, which was ∼174,000-fold higher than that of the parent strain PmCQ2, whose LD50 was 3.43 × 103 CFU (8 CFU), the survival rates (58.3%) of mice infected with PmCQ2Δ0442 was significantly increased  by intramuscular injection. Mice were challenged with approximately 3.08 × 107 CFU PmCQ2 at day 21 after the immunization. In the immunized group, 70–80% of the mice survived after challenge, while all control mice were dead within 1 week  of PmCQ2 and PmCQ2Δ0442 by transcriptome analysis in vitro and in vivo. A total of 394 DEGs were observed, including 152 up-regulated and 242 down-regulated genes in vitro (Fold change ≥ 1.5)   . GO and e ≥ 1.5) . Capsulee ≥ 1.5) , and LPSe ≥ 1.5) , iron ute ≥ 1.5)  and TonBe ≥ 1.5)  related ecreased . Similare ≥ 1.5) . TonB-dee ≥ 1.5) . The abo–/– macrophages, but not TLR4–/– macrophages  , hyaBCDE (capsule biosynthesis), and hexABCD (capsule export)  . There acharides , but themponents . The imp in mice . Here we export) . RNA seq reduced . Fis proed genes . HoweverP. multocida , VipA (Vi polysaccharide biosynthesis protein), galE and capD (lipopolysaccharide biosynthetic process) were significantly down-regulated in PmCQ2Δ0442. Moreover, the LPS content was also sharply reduced. But the mechanism by which Pm0442 affects LPS synthesis-related genes is still unclear.Lipopolysaccharide is also recognized as one of the most important factors associated with the pathogenesis of ultocida . The LPStructure . TranscrP. multocida constitute the major components of the bacterial cell surface, which play key roles in a range of interactions between the bacteria and the hosts. Both polysaccharides are involved in resistance to phagocytosis, they are therefore essential for virulence  and iron regulation/acquisition related genes  were markedly reduced in PmCQ2Δ0442 based on RNA sequencing. TbpA gene encodes transferrin-binding protein A that is important for iron uptake in Pasteurellaceae  system  of the total genes in the Pm70 genome encode proteins involved in iron uptake or acquisition . Similarellaceae . The afueumoniae . The fec) system . The hemeumoniae . The proparasuis . The Sitmolytica . Study hultocida , but the–/– macrophages compared to WT macrophage. This result indicates that TLR2 is also involved in the secretion of inflammatory factors in macrophages infected by PmCQ2.Innate immune responses play important roles in host defense against bacterial infection. As an important component of the mammalian natural immune system, TLRs are expressed in immune cells such as macrophages, dendritic cells (DCs) and B lymphocytes . Our preMycobacterium tuberculosis, OspA of Borrelia burgdorferi, and U-OMP19 and U-OMP16 of Brucella abortus can activate TLR2 only in the presence of acylation  to mediate pro-inflammatory cytokine secretion of IL-1β, TNF-α, IL-6, and IL-12p40 in macrophages via activation of the NF-κB, ERK1/2 and p38 signaling pathways. Notably, PmCQ2Δ0442 is a potential vaccine candidate strain. Our findings demonstrate that Pm0442 is a virulence-related gene that affects other virulence-related gene expression, and PM0442 protein directly binds to TLR2 to induce inflammatory responses of mice, which provides new guidance for the prevention and control of Pasteurellosis.In conclusion, https://www.ncbi.nlm.nih.gov/, PRJNA597831.The datasets generated in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The animal study was reviewed and approved by the Laboratory Animal Welfare and Ethical Commission of the Southwest University (Permit No. 11-1025), Chongqing, China.YP and NL designed the experiments. FH, XQ, NX, PL, XW, LD, and YD conducted the experiments. FH, XQ, RF, and NL analyzed the data and prepared the figures. FH, XQ, and PH drafted the manuscript. YP, RF, NL, and PH revised and approved the final manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Diffuse large B‐cell lymphoma (DLBCL) is an aggressive subtype of lymphoma, and multiple extranodal involvement (ENI) indicates adverse clinical outcomes. The aim of this study was to investigate the influence of oncogenic mutations and tumor microenvironment alterations on ENI in DLBCL.The clinical features of 1960 patients with newly diagnosed DLBCL were analyzed, and DNA and RNA sequencing was performed on 670 and 349 patients, respectively. Oncogenic mutations and tumor microenvironment alterations were compared according to ENI and evaluated in zebrafish patient‐derived tumor xenograft models.MYD88 was frequently mutated in patients with multiple ENI, co‐occurred with mutations in CD79B, PIM1, TBL1XR1, BTG1, MPEG1, and PRDM1, and correlated with invasion of the bones, kidney/adrenal glands, breasts, testes, skin, and uterus/ovaries. For tumor microenvironment alterations, patients with multiple ENI showed higher regulatory T‐cell (Treg)‐recruiting activity, but lower extracellular matrix‐encoding gene expression, than those without ENI and with single ENI. Elevated Treg‐recruiting activity was related to mutations in B2M, SGK1, FOXO1, HIST1H1E, and ARID1A, and correlated with invasion of the bone marrow and thyroid. Additionally, mutations in MYD88, PIM1, TBL1XR1, SGK1, FOXO1, HIST1H1E, and ARID1A were associated with decreased major histocompatibility complex class I expression. Zebrafish models further revealed relationships between MYD88 mutations and invasion of the kidneys and gonads, as well as B2M mutations and invasion of the bone marrow. Increased CXCR4 expression is linked to bone marrow invasion in an organotropic way.Multiple ENI was significantly associated with poor performance status, advanced stage, elevated serum lactate dehydrogenase, low response rate, and inferior prognosis. Lymphoma invasion of the bones, spleen, bone marrow, liver, and central nervous system were independent unfavorable prognostic factors. Our findings thus contribute to an improved understanding of the biological behavior of multiple ENI and provide a clinical rationale for targeting ENI in DLBCL. 1. DLBCL patients with multiple extranodal involvement presented advanced disease stage and poor prognosis.2. Oncogenic mutations were associated with multiple extranodal involvement.3. Increased Treg‐recruiting activity and decreased extracellular matrix expression of the tumor microenvironment correlated with multiple extranodal involvement. CHTcaudal hematopoietic tissueCNScentral nervous systemCTcomputed tomographyDLBCLdiffuse large B‐cell lymphomaECMextracellular matrixECOGEastern Cooperative Oncology GroupENIextranodal involvementFFPEformalin‐fixed paraffin‐embeddedGSEAgene set enrichment analysisIPIInternational Prognostic IndexLDHlactate dehydrogenaseMDSCmyeloid‐derived suppressor cellMRImagnetic resonance imagingNCCNNational Comprehensive Cancer NetworkNF‐κBnuclear factor kappa‐Bnon‐GCBnon‐germinal center B cellOSoverall survivalPET‐CTpositron emission tomography‐computed tomographyPFSprogression‐free survivalR‐CHOPrituximab, cyclophosphamide, doxorubicin, vincristine, and prednisoneSNVsingle nucleotide variationSPSSStatistical Package for the Social Sciences.Th2T helper 2TIPtumor immunophenotypingTregregulatory TWESwhole exome sequencingWGSwhole genome sequencingWHOWorld Health Organization1Diffuse large B‐cell lymphoma (DLBCL) is the most common histological subtype of aggressive non‐Hodgkin lymphoma and represents a heterogeneous entity.MYD88/CD79B‐mutated genotype. In contrast, gastric and thyroid DLBCL lack MYD88 mutations.MYD88/CD79B mutations, including PIM1, BTG1, TBL1XR1, MPEG1, and PRDM1, were frequently acquired in breast, testis, and cutaneous lymphoma.Accumulating data suggest the occurrence of divergent biological behaviors in extranodal DLBCL. For instance, DLBCL in immune‐privileged sites, including the CNS and testes, as well as breast, uterine, and cutaneous DLBCL, are related to prevalent non‐germinal center B cell (non‐GCB) phenotype and In the present study, we investigated the clinical characteristics and prognostic significance of multiple ENI in a large cohort of 1960 patients with newly diagnosed DLBCL, and performed genomic and transcriptomic analyses to illustrate the oncogenic mutations and tumor microenvironment alterations associated with ENI.22.1A flow chart is outlined in Figure Clinical data included age, gender, Eastern Cooperative Oncology Group (ECOG) performance status, Ann Arbor stage, serum lactate dehydrogenase (LDH), and specific sites of ENI.2.2http://genome.ucsc.edu). The Refseq database (Human Reference Genome version hg19) was used as the reference genome. The filtration of detected SNVs and indels was performed by homemade pipeline with the above software. Details for DNA sequencing are provided in the Supporting Information Methods.Genomic DNA was extracted from frozen tumor tissue by a QIAamp DNA Mini Kit , or from formalin‐fixed paraffin‐embedded (FFPE) tumor tissue by a GeneRead DNA FFPE Tissue Kit (Qiagen), based on the manufacturer's guidelines. For 216 patients, whole‐exome sequencing (WES) was carried out on frozen tumor tissue (n = 132), or on FFPE tumor tissue quality controlled by agarose gel electrophoresis (n = 84). For 109 patients, whole‐genome sequencing (WGS) was performed on frozen tumor tissue. WES  and WGS (n = 17) were performed on 42 matched peripheral blood samples randomly selected to build a somatic mutation calling principle and to exclude germ‐line polymorphisms. Three hundred forty‐five patients with FFPE tumor tissue were analyzed by targeted sequencing of 55 lymphoma‐associated genes. Genome Analysis Toolkit , Haplotype Caller, and GATK Unified Genotyper were applied to call single nucleotide variations (SNVs) and indels, which were mapped to the genome location using the UCSC Genome Browser . RNA quantity was assessed on Nanodrop, and the integrity of total RNA was estimated by RNA 6000 Nano Kit on Aligent 2100 Bioanalyzer. Among the 670 patients with DNA sequencing data, RNA sequencing was performed on qualified frozen tumor tissue from 349 patients. Read pairs were aligned to Refseq hg19 by Burrows‐Wheeler Aligner version 0.7.13‐r1126. The HTSeq was applied to generate transcript counts table files.2.4https://doi.org/10.5281/zenodo.3700087) as described by Wright et al.DLBCL genotypes were identified using the LymphGen probabilistic classification tool . Pathways were considered statistically significant when the P value was <.05, and the false discovery rate was <.25.GSEA results were presented as the upregulation or downregulation of the desired gene set using GSEA v4.0.1 software and Molecular Signature Database.2.6http://biocc.hrbmu.edu.cn/TIPThe activity score of anti‐tumor immunity was generated using tracking tumor immunophenotyping , HLA‐B , HLA‐C , and CXCR4 . Immunohistochemistry of zebrafish model tissues was performed on 3 μm agarose sections,For immunofluorescence, zebrafish model tissues were fixed in 4% formaldehyde and stored in methanol at −20°C. Tumor cells were labeled by Dil, and nuclei were counterstained with DAPI. Pictures were captured in a Zeiss880 microscope.2.9χ2 test or Fisher's exact test. Differences of immunity activity scores and normalized gene expression in two groups were analyzed using Mann‐Whitney U test. Progression‐free survival (PFS) was measured from the date of diagnosis to the date when disease progression/relapse was recognized or the date of last follow‐up. Overall survival (OS) was calculated from the date of diagnosis to the date of death or the date of last follow‐up. Survival functions were generated with the Kaplan‐Meier method and compared by the log‐rank test. Univariate hazard was estimated using the Cox regression method. Significant variables on univariate analysis were kept in multivariate set. Statistical significance was defined as P < .05. All P values in this manuscript were reported without mathematical correction. The above statistical analyses were performed by Statistical Package for the Social Sciences (SPSS) 26.0 software .The best cutoffs of continuous variables were obtained by maximizing the Youden's index using receiver operating characteristic (ROC) curve analysis. Baseline characteristics of patients were ascertained using Pearson's 33.1P < .0001), advanced Ann Arbor stage (P < .0001), elevated serum LDH (P < .0001), as well as increased prevalence of the non‐GCB subtype (P = .0059) compared to those without ENI and with single ENI. No significant differences in Epstein‐Barr virus‐encoded RNA positivity  or the proportion of double/triple‐hit lymphomas indicating BCL2/BCL6/MYC rearrangements  were observed between patients with multiple ENI and those without ENI and with single ENI. Meanwhile, patients with multiple ENI showed a remarkable decrease in overall response rate (75.5%) and 2‐year OS rate (61.8%) relative to those without ENI  and with single ENI  following R‐CHOP treatment.A total of 1960 patients with newly diagnosed DLBCL were analyzed, including 662 patients without ENI, 902 patients with single ENI, and 396 patients with multiple ENI. The clinical characteristics of the patients are summarized in Table According to specific extranodal organs, DLBCL was observed most often in the GI tract (27.1%), followed by the bones (11.4%), spleen (11.0%), bone marrow (8.0%), kidney/adrenal glands (5.5%), lungs (5.3%), breasts (4.8%), liver (4.0%), pancreas (3.5%), testes (3.3%), CNS (3.0%), thyroid (2.5%), skin (1.9%), nasal cavity (1.6%), and uterus/ovaries  were independent prognostic factors of inferior PFS and OS when adjusted by standard prognostic factors, including age , performance status , Ann Arbor stage , and serum LDH . Using univariate analysis, lymphoma invasion of the bones, spleen, bone marrow, kidney/adrenal glands, lungs, liver, and CNS were unfavorable predictors of both PFS and OS, while lymphoma invasion of the GI tract was related to favorable outcomes Figure . Signifi3.2MYD88 mutations were significantly increased in patients with multiple ENI (22.2% or 32/144) compared to those without ENI and with single ENI . The MYD88L265P mutation, either alone (18.8% or 27/144) or with the CD79B mutation (4.2% or 6/144), was more frequently observed in patients with multiple ENI than those without ENI and with single ENI . As revealed by GSEA , and B‐cell receptor signaling pathway was upregulated according to MYD88L265P/CD79B double mutation (P = .0204).Oncogenic mutations closely related to DLBCL were analyzed in 670 patients, including 216 cases by WES, 109 cases by WGS, and 345 cases by targeted sequencing. As shown in Figure MYD88 mutations also co‐occurred with mutations in PIM1, TBL1XR1, BTG1, MPEG1, and PRDM1. Among the 105 patients with MYD88 mutations, 53 patients (50.5%) had a PIM1 mutation, 22 patients (21.0%) had a TBL1XR1 mutation, 22 patients (21.0%) had a BTG1 mutation, 15 patients (14.3%) had a MPEG1 mutation, and 14 patients (13.3%) had a PRDM1 mutation. Correlations between MYD88‐related genes included PIM1 and BTG1 (34 cases), PIM1 and TBL1XR1 (28 cases), PIM1 and MPEG1 (24 cases), TBL1XR1 and MPEG1 (11 cases), BTG1 and MPEG1 (11 cases), and MPEG1 and PRDM1 , kidney/adrenal glands , breasts , testes , skin , and uterus/ovaries  compared to those without invasion of the bones (14.1% or 82/581), kidney/adrenal glands (14.9% or 95/637), breasts (13.8% or 86/625), testes (15.0% or 98/654), skin (15.0% or 97/647), and uterus/ovaries (15.3% or 101/662). PIM1 mutations were higher in patients with breast invasion than those without invasion . TBL1XR1 mutations were higher in patients with testes invasion than those without invasion . MPEG1 mutations were higher in patients with bone invasion than those without invasion .TET2 mutations  but decreased mutations in DUSP2 , MPEG1 , and HIST1H1E , MYD88 , and PIM1  were observed in patients with GI tract invasion compared to those without invasion . Mutations in KMT2D , SOCS1 , BCL6 , and IRF8  were higher in patients with spleen invasion than those without invasion . Mutations in FAS  and NFKBIE  were higher in patients with lung invasion than those without invasion . Mutations in BCL6  were higher in patients with liver invasion than those without invasion .Genetic aberrations related to other prognosis‐related extranodal organs are shown in Figure S2. Increased P = .0021; Figure P = .0002 and P = .0187, respectively; Figure One hundred twenty‐three (37.9%) of the 325 patients were genetically classified . MCD genotype was more frequently observed in patients with multiple ENI than those without ENI and with single ENI , while other immune cell subsets showed no significant differences, including natural killer cells, dendritic cells, macrophages, CD8+ T cells, CD4+ T cells, Th1 cells, Th2 cells, Th17 cells, and MDSCs. All patients were subsequently divided into two groups, Treg‐high (n = 89) and Treg‐low (n = 260), according to the optimal cutoff estimated by ROC curve analysis. Gene ontology analysis revealed that T‐cell immunity , chemokine signaling , and other immune‐associated signaling  were downregulated in Treg‐high patients compared to Treg‐low patients , SGK1 , FOXO1 , HIST1H1E , and ARID1A  and thyroid  and proteoglycans/glycoproteins , as well as genes encoding endothelial cells , were screened using RNA sequencing data. Patients with multiple ENI presented decreased expression levels of genes encoding collagens and proteoglycans/glycoproteins compared to those without ENI and with single ENI  and HIST1H1E . Similarly, decreased HLA‐B expression and reduced positivity of HLA‐B expression were significantly associated with mutations in MYD88 , PIM1 , and TBL1XR1 . Decreased HLA‐C expression and reduced positivity of HLA‐C expression were significantly associated with mutations in FOXO1  and ARID1A .The influences of ENI‐related mutations on MHC class I expression were evaluated. Decreased 3.5CXCR4 expression was associated with bone marrow invasion . Increased percentages of kidney invasion  and gonad invasion  were observed in zebrafish models with MYD88 mutations compared to those without mutations  was assessed by immunofluorescence. Representative images of lymphoma invasion in organs are shown in Figure s Figure . With re4Multiple ENI is closely related to high tumor burden that manifests from poor performance status, advanced disease stage, and elevated serum LDH, leading to poor response to rituximab‐containing immunochemotherapy and adverse clinical outcomes in DLBCL. Our data displayed an increased prevalence of GI tract involvement as compared to the previous report.MYD88),BCL6, IRF8, KMT2D, and SOCS1),FAS and NFKBIE),BCL6),MYD88, PIM1, MEPG1, DUSP2, and HIST1H1E).Based on a large patient cohort and genomic data, we revealed the divergent survival status and genetic alterations for DLBCL in terms of specific extranodal organs. Clinically, in addition to the bone marrow, CNS, liver, and lungs defined by NCCN‐IPI,MYD88 is implicated in tumorigenesis through proinflammatory mechanismsMYD88 mutations and multiple ENI, indicating another important biomarker of ENI in DLBCL. Dysregulation of the P53 and B‐cell receptor signaling pathways was present in MYD88‐mutated DLBCL, which resulted in constitutive activation of nuclear factor kappa‐B (NF‐κB) pathway, resistance to chemotherapy, but sensitivity to the BTK inhibitor.MYD88, including PIM1, TBL1XR1, BTG1, MPEG1, and PRDM1, all of which presented high frequency in the MCD subtype defined by Schmitz et al, contributing to NF‐κB activation in a B cell receptor‐dependent manner.PIM1TBL1XR1MYD88L265P alone is insufficient to drive malignant transformation in B cells and may cooperatively induce lymphoma with other genetic events.Among oncogenic mutations, B2M has recently been identified in the immune regulation of Treg cells.SGK1‐FOXO1 signaling is essential for Treg cell migration.H1ST1H1E and ARID1A in immune cells have not been fully identified, changes mediated by epigenetic regulations may alter the function of T cells in anti‐tumor immunity.Tumor microenvironment plays a critical role in DLBCL progression.SGK1 and HIST1H1E, HLA‐B with mutations of MYD88, PIM1, and TBL1XR1, and HLA‐C with mutations of FOXO1 and ARID1A, indicating possible role of alterations in MHC class I expression on immune surveillance by anti‐tumor T cells in DLBCL.MHC class I expression is critically involved in the process of immune recognition.To further explain the organotropic dissemination of DLBCL, we analyzed the expression of chemokine receptors in the tumor microenvironment, which promote the migration of malignant B cells,5Extranodal DLBCL is heterogeneous in clinical and molecular features, represented by variable oncogenic mutations and tumor microenvironment alterations according to specific extranodal sites. Thus, further prospective clinical studies are necessary to establish biologically driven therapeutic strategies against ENI in DLBCL.Rong Shen and Peng‐Peng Xu performed the experiments, collected and analyzed the data, and wrote the article. Nan Wang prepared biological samples and performed the experiments. Rong Shen, Peng‐Peng Xu, Nan Wang, Hong‐Mei Yi, Lei Dong, Di Fu, Jin‐Yan Huang, and Heng‐Ye Huang recruited patients, collected study data, and prepared biological samples. Hong‐Mei Yi and Lei Dong reviewed the histopathologic diagnoses. Di Fu and Jin‐Yan Huang carried out the sequencing and participated in the validation experiments. Rong Shen and Di Fu were responsible for bioinformatics investigation. Peng‐Peng Xu gave technical support. Heng‐Ye Huang was responsible for statistical review. Anne Janin, Shu Cheng, and Li Wang gathered detailed clinical information for the study. Wei‐Li Zhao conceived the study, directed and supervised research, and wrote the manuscript.The study was approved by the Shanghai Ruijin Hospital Review Board. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki. All tissues used for zebrafish patient‐derived tumor xenograft model establishment were obtained from Shanghai Ruijin Hospital with written informed consent. The study was approved by the Ethics Committees of Shanghai Ruijin Hospital. All experimental procedures followed the rules of the Committee on Animal Care of Shanghai, China. All animal experiments were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University.The authors declare that they have no conflict of interest.FIGURE S1 Standard GSEA graphs for the p53 signaling pathway (upper panel) and B‐cell receptor pathway (lower panel) in MYD88 mutations (A), MYD88L265P mutations alone (B), or with CD79B mutations (C)Click here for additional data file.FIGURE S2 Oncogenic mutations (A‐D), molecular classification (E and F) and tumor microenvironment alterations (G) related to prognosis‐related organs. P values comparing different percentages are indicated in subgroupsClick here for additional data file.FIGURE S3 Normalized gene expression by RNA sequencing and positivity of HLA expression by immunohistochemistry (x400 magnification) for HLA‐A (A), HLA‐B (B), and HLA‐C (C) on patient tumor samplesClick here for additional data file.Supplementary informationTABLE S1 Extranodal involvement of patients evaluated by PET‐CT and CT/MRIClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationSupplementary methods.Click here for additional data file.