Characterization Of The Host Immune Responses In Mice After Inhaling Aerosols Of Hypervirulent Klebsiella Pneumoniae

Klebsiella pneumoniae(KP)is a Gram-negative pathogen,which is easy to colonize in human respiratory tract and intestine.At present,the most popular pathotypes are classic Klebsiella pneumoniae(c KP)and hypervirulent Klebsiella pneumoniae(hvKP).c KP is a opportunistic pathogen that usually infects people,who are immunocompromised,or who have existing barrier breakdown.hvKP produced more capsular polysaccharides than c KP has ability to infect healthy individuals.It is easy to cause a variety of clinical symptoms and endanger the life of the patient.In recent years,the boundary between these two strains has become increasingly blurred,and reports of hypervirulent and multidrug-resistant strains are on the rise.For example,in 2016,five surgical patients in The Second Affiliated Hospital of Zhejiang University successively contracted ST11 carbapenease-producing hvKP,during their stay in the intensive care unit,and eventually died of severe pneumonia,multiple organ failure and sepsis due to treatment failure with a variety of antibiotics.With the spread and increased detection rate of such strains,the bacteria will seriously endanger the life and health of patients,and pose an unpredictable threat to public health.Therefore,this study intends to characterize the inhalational toxicology and host immune response of hvKP,so as to develop vaccines and drugs for effectively controlling the infection and spread of hvKP and multidrug-resistant hypervirulent Klebsiella pneumoniae(MDR-hvKP).In this study,a mouse model infected with hvKP was established via aerosolized intratracheal inoculation(i.t.).Different doses of liquid aerosols of hvKP strain NTUH-K2044 were delivered to the lungs of C57BL/6J mice by the Micro Sprayer Aerosolizer.A median lethal dose(LD₅₀)was calculated to be about 200CFU according to the survival curve.Then 100×LD₅₀hvKP were administered to mice.And the clinical observation showed that a large number of mice died on the third day after challenge.The histopathology results showed that there was a large number of neutrophil infiltration around bronchioli terminales in the lungs of mice 12h after hvKP challenge,and the infiltration intensified with time.By 60h,alveolar spaces was filled with mucus,and partial alveolar walls was broken.It was found in spleen that lymphocytes in the medulla were significantly reduced at 36h after infection,and the proliferation of lymphoid cells under the capsule was seen at 60h.In addition,there were no obvious pathological changes in heart,liver and kidney.At last the bacterial load in organs were tested,and the results showed that the bacteria in the lungs began to increase at 6h after challenge,and then increased exponentially.Hv KP colonies were detected in the liver36h after challenge and in blood,heart,spleen and kidney 48h after challenge.From the above results,it can be seen that the inhalational model of Hv KP exposure to mouse was successfully constructed.Subsequently,the dynamic changes of the cytokines and immune cells in infected mice were studied,using luminex liquid chip technology and flow cytometry.A variety of cytokines in serum and bronchoalveolar lavage fluid(BALF),as well as cell populations in BALF were tested after 100×LD₅₀ hvKP challenging.The results showed that levels of GM-CSF,GRO-?,TNF-?,IP-10,MIP-1?,IL-6 and MIP-2 increased significantly at 6h after infection,and the levels of IL-9,G-CSF,IL-1?,RANTES,LIF,IL-22,IFN-?and IL-17A increased significantly at 24h after infection.Cytokines in serum increased mainly at 48 h after infection,and the increases include IL-9,G-CSF,IL-18,IL-10,GRO-?,TNF-?,IP-10,MIP-1?,IL-6,MIP-2.The proportion of lung neutrophil increased at 12h and became stable at 24h.After infection,the proportion of alveolar macrophage(AMs),Natural killer(NK)cell,B cells and T cells all decreased.In addition,the proportion of interstitial macrophages(IMs),dendritic cells(DCs),epithelial cells,and endothelial cells did not change significantly.These results suggest that there was a severe inflammatory reaction in the lungs of mice.Then,trend of the changes of main cell groups and intracellular gene expression in lung were analyzed at the transcriptome level,using the single-cell RNA sequencing(scRNA-seq)technologies.The results showed that the number of neutrophils and monocytes increased rapidly from 12h after infection,and the number of monocytes remained stable at 48h after infection,the number of neutrophils remained increased until 60h after infection;The number of AMs increased after infection and began to decrease at 48h;the number of B,T and NK cells decreased after infection,while DC cells increased at first and then decreased.In-depth analysis of significantly changed proteins tested by luminex liquid chip technology,it was found that IL-18 is mainly expressed in AMs and mesenchymal cells,and the highest expression level was 48h after infection;IP-10 was found expressed highly in a variety of cells,especially in monocytes,mesenchymal cells and IMs,and the expression level reached the peak at24h;GM-CSF was mainly expressed in epithelial cells,the expression level was low,but the overall trend was increasing with infection;TNF-?was expressed by neutrophils,monocytes,and macrophages.Alveolar macrophages had the highest expression level who reached the peak at 48 h after infection;the expression level of IL-17 was low and mainly concentrated in the epithelium and T cells;IL-10 mainly expressed in neutrophils and monocytes,and reached to peak at 60 h after infection;IL-6 increased with infection and mainly expressed in fibroblasts and mesenchymal cells;IL-1?showed a gradual increase trend,and was secreted by all kinds of cells,and the higher expressed level was found in neutrophils,monocytes,Macrophages and DCs.Finally,the data from the transcriptome,cell and protein testing level were analyzed comprehensively.The result revealed that the gene expression trends of GRO-??IL-18?MIP-1??MCP-1?IP-10?GM-CSF?TNF-??IL-6?IL-17?IL-10?IL-1?were consistent with these protein expression trends.In addition,the trends in neutrophil,AM,NK,B and T cells were consistent at the level of cellular and transcriptome.To sum up,this study had successfully established a mouse model infected with hvKP aerosols.The trend of the changes of various cytokines of the model was systematically demonstrated by protein test and gene expression test.The changing trends and functions of the innate immune cells were roundly described.This work provided a detailed data for revealing the mechanism of hvKP treatment.At the same time,it also laid the foundation for the follow-up solution to the infection of MDR-hvKP.