| Objective: Aspergillus fumigatus(A.fumigatus)is one of the most common and opportunistic aerial fungal pathogens,which can cause fatal invasive aspergillosis in immunocompromised patients.Diabetes has become a risk factor for the development of aspergillosis infection,even greater incidence of invasive pulmonary aspergillosis was found in diabetic patients than in immunocompromised patients.In hyperglycemic state,the infection could elevate the level of proinflammatory cytokines,the latter in turn causes insulin resistance.These lead to a vicious cycle and aggravate the infection severity.Hypoxia usually forms as a microenvironment at sites of A.fumigatus infection.As a core factor of hypoxia environment,the transcription factor hypoxia inducible factor-1α(HIF-1α)is activated and plays an important role of anti-fungal immune and inflammatory responses.Many researches have shown that HIF-1α was suppressed and dysfunction in hyperglycemic state.Therefore,the inhibitory effect of HIF-1α is likely to be an important factor leading to exacerbation of infection.But the mechanism is still unclear.There has been evidence that HIF-1α played an important role in regulating immunity and inflammation,and it can effectively help the host defend against infection.However,the role of HIF-1α signal transduction in A.fumigatus infection in diabetes is still unknown.The purpose of this research is to explore the characteristics of infection through clinical retrospective study and the mouse model of pulmonary A.fumigatus infection combined with diabetes.And we also explored the role of HIF-1α in pulmonary A.fumigatus infection combined with diabetes and its mechanism.These in turn provide a new theoretical basis for the therapy of diabetic patients with pulmonary A.fumigatus infection.Methods: 1.The infection characteristics and the expression of HIF-1α in pulmonary A.fumigatus infection combined with diabetes.In this part of the experiment,firstly,we performed a clinical retrospective study of patients with fungal pneumonia in the First Hospital of China Medical University from March 2008 to July 2018,and analyzed the impact of diabetes on the course of infection.In animal models,all experiments were carried out using SPF adult male C57/bl6 mice.All animals were randomly divided into normal control group(nondiabetic group,n = 30),normal infection group(nondiabetic,A.f group,n = 30),diabetic control group(diabetic group,n = 30)and the diabetic infection group(diabetic,A.f group,n = 30).According to previous researches,a single high-dose intraperitoneal injection of streptozotocin(STZ)was used to establish murine model of diabetes.Using tracheal intubation technique,mice were inoculated with 50 microliters of A.fumigatus spore suspension or sterile PBS through the trachea.The mice of each group were sacrificed on day 1,2,3,4,7,14 after intubation.Infection status was assessed by survival rate,lung homogenate colony counts,immunofluorescent staining of A.fumigatus spores in the lung,and serum A.fumigatus antigen ELISA test.The inflammatory response was evaluated by HE staining of lung tissue pathological sections to observe leukocyte infiltration and serum cytokines detection.The expression of HIF-1α was detected by immunoblotting of lung tissue.2.The role of HIF-1α in diabetic mice of pulmonary A.fumigatus infection and its mechanismAll experiments were carried out using SPF adult male C57/bl6 mice in Section Two.The mice were randomly divided into normal group(nondiabetic group,n = 30),diabetes group(diabetic,vehicle group,n = 30),and diabetes therapy group(diabetic,DMOG group,n = 30).According to previous researches,a single high-dose intraperitoneal injection of streptozotocin(STZ)was used to establish murine model of diabetes.All mice were inoculated with 50 microliters of A.fumigatus spore suspension via trachea using tracheal intubation technique.The diabetic,DMOG group activated HIF-1α by intraperitoneal injection of dimethyloxalylglycine(DMOG).The mice of each group were sacrificed on day 1,2,3,4,7,14 after intubation.Infection status was assessed by survival rate,lung homogenate colony counts,immunofluorescent staining of A.fumigatus spores in the lung,and serum A.fumigatus antigen ELISA test.The inflammatory response was evaluated by HE staining of lung tissue pathological sections to observe leukocyte infiltration and serum cytokines detection.The expression of HIF-1α was detected by immunoblotting of lung tissue.The possible mechanism of HIF-1α was explored by RNA sequencing of the lung tissue.3.Effects of hyperglycemia on HIF-1α activation in human THP-1 macrophages induced by A.fumigatus spores and its downstream signaling pathway in hypoxia conditionIn this experimental part,we divided the macrophages induced by human THP-1 cells into 5.5mM glucose concentration group,30 mM glucose concentration group,30 mM + DMOG group,30 mM + DMOG + Alum group.The agonist of HIF-1α,DMOG,and NLRP3 inflammasome agonist aluminum hydroxide(Alum)were used to modulate the expression of HIF-1α and NLRP3 inflammasome,respectively.Macrophages were cocultured with A.fumigatus spores in hypoxia conditions,and then macrophages and cell culture supernatants were collected at 0,12,and 24 hours,respectively.The expression of HIF-1α and NLRP3 were detected by immunoblot of macrophages.The secretion of IL-1β in the cell supernatant was detected by ELISA.Naive T cells were added and cocultured with A.fumigatus-activated macrophages for 48 hours.Flow cytometry was used to detect nuclear,intracellular,and surface factors,and to evaluate the differentiation ratio of helper T cells(Th cells)and regulatory T cells(Treg cells).Results: 1.The infection characteristics and the expression of HIF-1α in pulmonary A.fumigatus infection combined with diabetes.1.1 The clinical retrospective study found that in fungal pneumonia patients,diabetic patients had longer hospital stays compared with non-diabetic patients.Immune deficiency and diabetes can be independent risk factors for prolonged hospital stay in patients with fungal pneumonia.(P <0.05)1.2 Compared with other groups,the mortality of mice in the diabetic infection group was significantly increased,and the mice were mainly dead in the first three days after infection.(P <0.05)1.3 Compared with the nondiabetic,A.f group,the spore clearance of the mice in the diabetic,A.f group was slower.By the 14 th day after infection,the lung homogenate colony count and A.fumigatus antigen immunofluorescence detection were still positive.The expression of serum A.fumigatus antigen in the diabetic,A.f group was also higher than that in the nondiabetic,A.f group.(P <0.05)1.4 Compared with the nondiabetic,A.f group,the inflammatory response of the mice in the diabetic,A.f group was more severe.After infection,the lung of the diabetic,A.f group showed diffuse inflammatory cell infiltration and delayed regression.The release of inflammatory cytokines in serum was generally elevated,reaching a peak on day 2 or 3 after infection.(P <0.05)1.5 After infection,HIF-1α was activated in the lungs of nondiabetic,A.f group(P <0.05),while HIF-1α expression in the diabetic,A.f group was not significantly different from that in the diabetic group.2.The role of HIF-1α in diabetic mice of pulmonary A.fumigatus infection and its mechanism 2.1 DMOG can activate HIF-1α in the lung tissue of diabetic mice of pulmonary A.fumigatus infection.(P <0.05)2.2 After the application of DMOG,the mortality of diabetic mice decreased,and spore clearance of A.fumigatus was accelerated.By the 14 th day after infection,the lung homogenate colony count and A.fumigatus antigen immunofluorescence detection had turned negative.The peak of A.fumigatus antigen expression in serum also decreased.(P <0.05)2.3 After the application of DMOG,the inflammatory response in diabetic mice was alleviated.The infiltration of leukocytes has reduced and can resolve on time.On day 2 after infection,inflammatory cytokines released in the serum was generally reduced compared with vehicle group.(P <0.05)2.4 RNA sequencing showed that compared with the nondiabetic group,the expression of genes related to inflammation in diabetic group was significantly elevated,and multiple inflammation-related signal pathways were activated,and the application of DMOG can correct the excessive activation of the inflammatory response by activating HIF-1α.(P <0.05)3.Effects of hyperglycemia on HIF-1α activation in human THP-1 macrophages induced by A.fumigatus spores and its downstream signaling pathway in hypoxia condition 3.1 In hyperglycemia,after stimulated by A.fumigatus spores in hypoxia condition,HIF-1α in macrophages was not activated in time,NLRP3 inflammasome was over-activated,and IL-1β secretion was also increased.(P <0.05)3.2 After DMOG application,HIF-1α can be activated in hyperglycemic condition,and excessive activation of NLRP3 and IL-1β can be corrected(P <0.05).Alum,an agonist of NLRP3 inflammasome,can only increase the expression of NLRP3 and IL-1β,but has no effect on the expression of HIF-1α.3.3 In hyperglycemic condition,A.fumigatus-activated macrophages can induce T cells to differentiate into Th2 and Th17 in hypoxia condition,while the differentiation ratio of Th1 and Treg decreased(P <0.05).DMOG can correct this bias of Th and Treg cell differentiation(P <0.05),but the application of Alum could dampen the regulation of DMOG.Conclusions: 1.Diabetes aggravated pulmonary A.fumigatus infection.The main manifestations were high mortality,slow spore clearance,and severe inflammatory response.2.When pulmonary A.fumigatus infection combined with diabetes,the activation of HIF-1α was inhibited.3.Stabilization of HIF-1α can alleviate the pulmonary A.fumigatus infection,reduce mortality,accelerate spore clearance,and inhibit excessive inflammatory responses.4.In hyperglycemic hypoxia condition,HIF-1α may regulate T cell differentiation by regulating the activation of NLRP3 inflammasome,and then exert its protective effect against A.fumigatus infection. |
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