The Role Of NK Cells In The Pathogenesis Of Brain Injury Caused By Angiostrongylus Cantonensis Infection

Angiostrongylus cantonensis(A.cantonensis),a rat lung nematode,is a foodborne zoonotic parasite.It was first discovered in the pulmonary arteries and hearts of domestic rats in Guangzhou(Canton),China,by Chen in 1935.The first human case of angiostrongyliasis was reported in Taiwan in 1945.So far,nearly 3000 cases of the disease have been documented worldwide.A.cantonensis has spread from its traditional endemic regions of Southeast Asia and the Pacific islands to the American continent,Africa and Australia.Sporadic cases and outbreaks of human angiostrongyliasis have been frequently reported in China in recent years.Therefore,angiostrongyliasis has become a potentially fatal globally emerging infectious disease.Humans,non-permissive hosts of A.cantonensis,become infected by eating raw or undercooked intermediate hosts including snails or slugs,paratenic hosts such as prawns,or contaminated vegetables that contain the infective larvae of the worm.The larvae are neurotropic and they exclusively present in the central nervous system(CNS).The main clinical manifestation of human angiostrongyliasis is eosinophilic meningitis.The common clinical symptoms include headache,neck stiffness,paresthesia,vomiting,and nausea.In some serious cases,it even causes death.The treatment of this disease includes supportive treatment,combined with corticosteroid therapy to reduce inflammation.The use of anthelminthic remains controversial,as it may exacerbate the inflammatory reaction and neurological symptoms.However,the pathogenesis of angiostrongyliasis is not fully understood.Physical damage caused by the migration of larvae,and its inflammatory reaction,toxicity and immune response may be involved.Natural killer cells(NK cells)are an important cell subset of the innate immune system and can respond rapidly to a variety of insults with cytolytic activity and cytokine secretion.It was reported that NK cells could readily home to the CNS under an array of pathological circumstances.However,very little is known about whether these NK cells are simply passive migrants or actively participate in the CNS pathogenesis.In the case of angiostrongyliasis,whether and how NK cells play a role in the pathogenesis or protection in the brain injury remains elusive.In this study,we first constructed a mouse model with A.cantonensis infection and observed the proportion and quantity of NK cells accumulated into brain.Then,the phenotype and function of NK cells in the CNS were detected.The role of NK cells in the pathogenesis of brain injury induced by A.cantonensis was elucidated by NK cell blocking and adoptive transferring experiments.Finally,the origin of NK cells in brain after infection was analyzed and the key chemokines for recruiting NK cells to CNS in the microenvironment of brain injury were detected.The main results are as follows:1 NK cells accumulated into brain of mice infected with A.cantonensis.Mice and humans are both non-permissive hosts of A.cantonensis,and the pathogenic process is comparatively similar.In this study,mice were used as experimental model for A.cantonensis infection.A small number of invasive larvae were found in the brain at 10 dpi(days of post infection).Nerve injury and inflammatory of the brain were found at 14 dpi.From the 18 dpi to 22 dpi,neurological symptoms aggravated,the inflammation of brain intensified,the survival rate decreased,and the disease severities reached the peak.To investigate the role of NK cells in brain injury induced by A.cantonensis,we examined whether NK cells accumulated in the CNS after infection.The results of immunohistochemistry and flow cytometry showed that NK cells began to appear in brain at 14 dpi.The proportion and quantity of NK cells increased gradually with the extension of infection time until 22 dpi(22 dpi vs 0 dpi:17.47±6.11%vs 0.45±0.12%,P<0.001;1.32±0.36×105 vs 696.9±617.2,P<0.001).2 NK cells in brain of mice infected with A.cantonensis have elevated cytotoxicity and secretory ability.We further examined the phenotypic and functional changes of NK cells in brain of mice after infection with A.cantonensis.The results showed that the expression of activated molecule CD69(20.07±3.91%vs 31.57±3.35%;P<0.05),activated receptor NKp46(22.57±10.74%vs 82.40±4.46%,P<0.001)and NKG2D(3.05±3.44%vs 13.81 ±6.39%,P<0.05)on bNKs(NK cells in brain)was decreased compared with sNKs(NK cells in spleen)from uninfected mice,while the expression of inhibitory receptor NKG2A remained low.Besides,the cytotoxicity of bNKs against YAC-1 cells was enhanced compared with sNKs in uninfected mice(Effect cells:Target cells=20:1,19.37%vs 8.35%).Simultaneously,the expression of CD107a on bNKs was up-regulated(11.58±2.16%vs 2.91±0.50%,P<0.001),and the level of IFN-γsecreted by bNKs was also increased(44.14±2.21 pg/ml vs 18.78±2.06 pg/ml,P<0.001).It is suggested that the NK cells in brain may increase their cytotoxicity through perforin/granzyme pathway and the elevated level of IFN-γ production.3 NK cells play an esssential role in aggravating brain injury after A.cantonensis infection in mice.We designed NK cell blocking and adoptive transferring experiments to observe the effects of depletion and increase of NK cells on brain injury induced by A.cantonensis infection.The results showed that the survival rate of infected mice increased(91.33%vs 77.08%,P<0.05),weight loss and nerve injury alleviated(17.35± 1.38 g vs 15.61±1.1 g,P<0.05),and the inflammation of brain decreased after blocking NK cells(IL-1β 25.88±2.38 pg/mg vs 30.75±2.83 pg/mg,IL-6 12.88±1.23 pg/mg vs 15.08±1.13 pg/mg,TNF-α 57.17±4.20 pg/mg vs 64.68±4.34 pg/mg.P<0.05).On the contrary,the survival rate and body weight of infected mice did not significantly change after adoptive transfer of NK cells,but nerve injury and the inflammatory in brain aggravated(IL-1β 36.36± 1.89 pg/mg vs 30.75±2.83 pg/mg,IL-6 18.08±0.55 pg/mg vs 15.08±1.13 pg/mg,TNF-α 76.84± 1.21 pg/mg vs 64.68±4.34 pg/mg,P<0.05).Therefore,it is suggested that NK cells play a role in exacerbating brain injury after A.cantonensis infection.4 The accumulation of NK cells in brain originated from increased hematopoiesis of bone marrow and migration of peripheral NK cells after A.cantonensis infection.To elucidate the origin of NK cells in brain tissue,we examined the effects of A.cantonensis infection on the proportion and quantity of NK cells in spleen,peripheral blood and bone marrow of mice.The results showed that the proportion and quantity of NK cells in spleen decreased(18 dpi vs 0 dpi:2.48±0.18%vs 4.09±0.41%,P<0.01;0.49±0.15×106 vs 2.07±0.42×106,P<0.001).So did the NK cells in peripheral blood(3.51±0.62%vs 5.99±0.66%,P<0.01;5371±299 vs 5.05±2.48×106,P<0.001).However,the proportion of NK cells in bone marrow significantly increased(7.42±1.24%vs 2.36±0.36%,P<0.001)and the ratio of NK precursor cells to mature NK cells up-regulated after infection(0.57±0.10 vs 0.29±0.109,P<0.001).In addition,the proportion and quantity of spleen NK cells were negatively correlated with those of brain NK cells(r=-0.790,P<0.01;r=-0.846,P<0.01),while the proportion of marrow NK cells was positively correlated with brain NK cells(r=0.866,P<0.001).The accumulation of NK cells in brain probably originated from increased hematopoiesis of bone marrow and migration of peripheral NK cells after A.cantonensis infection.5 The chemokines CCL3,CX3CL1 and CXCL10 in the microenvironment of brain injury may play a role in the recruitment of marrow and peripheral NK cells to the CNS in A.cantonensis infection.In order to identify the key chemokines for recruiting NK cells to CNS,we detected the expression of chemokines and their receptors in the brain of A.cantonensis infected mice.The results showed that the expression of several chemokines up-regulated in brain after infection,including CCL3(18 dpi vs 0 dpi:3.56±0.27 pg/mg vs 2.58±0.35 pg/mg,P<0.001),CX3CL1(421.7± 122.2 pg/mg vs 112.6± 13.38 pg/mg,P<0.001)and CXCL10(706.6± 17.71 pg/mg vs 607.00±54.10 pg/mg,P<0.05).Simultaneously,the expression levels of CCR1,CCR4,CCR5(CCL3 receptor),CX3CR1(CX3CL1 receptor)and CXCR3(CXCL10 receptor)were also increased.These results suggested that the chemokines CCL3,CX3CL1 and CXCL10 in the microenvironment of brain injury may play a role in the recruitment of marrow and peripheral NK cells to the CNS in A.cantonensis infection.Taken together,our study demonstrated for the first time that a large number of NK cells accumulated in the CNS and aggravated the brain damage in the mice infected with A.cantonensis.In addition,we revealed the key chemokines for the recruitment of NK cells from peripheral tissue to brain.Our findings have enriched the understanding of the pathogenesis of angiostrongyliasis and highlight another research direction for therapeutic intervention in CNS disease.