Study On The Regulatory Mechanism Of Chronic Helminth Infection On Immunopathology Of Mice Co-infected With Plasmodium Berghei

Malaria,AIDS,and tuberculosis is known are the world’s three major public health threats,and the clinical symptoms of malaria includes fever,anemia,liver and spleen swelling,nausea,vomiting,orthostatic hypotension,and so on.Plasmodium falciparum is the deadliest of all human Plasmodium parasites with more than 400,000 people died each year.Parasitic co-infection refers to the presence of two or more parasitic infections in the same host.The ecological requirements of helminthes and malaria parasites for disease transmission are very similar,and helminth infections are common in the tropical and subtropical areas with malaria transmission,and they are highly overlapped in geographical distribution.Therefore,co-infection of helminthes and malaria parasites is common in these areas.Trichinella spiralis is a common caus,and the life cycle of T.spiralis is complex.However,so far there are few studies about the immunopathologic regulation of malaria by co-infection of T.spiralis.ObjectiveTo investigate the modulation of chronic Trichinella spiralis(Ts)infection on immunopathology of mice co-infected with Plasmodium berghei ANKA(PbA),and provide scientific basis for immunological control of malaria.Methods1.Sixty specific pathogen free female Kunming mice(6-8 weeks old,weighting 18-22 g)were randomly divided into 4 groups,and Ts infection was performed at 121 days prior to PbA infection.(ⅰ)Uninfected control group;(ⅱ)Ts group:mice were mono-infected with 30 T.spiralis larvae by oral gavage on day 0;(ⅲ)PbA group:mice were mono-infected with 1×106 PbA-infected red blood cells in 0.1 ml of PBS administered by intraperitoneal injection;and(ⅳ)co-infected group(Ts+PbA):mice were infected with 30 T.spiralis and 121 days later challenged with 1×106 PbA.There were 15 mice in each group and 10 mice in each group were monitored for the mortality.The peripheral blood parasitemia of PbA group and Ts+PbA group was monitored daily by light microscope examination of Giemsa-stained thin tail-blood smears from day 3 after PbA infection.Mice were sacrificed on day 135 after Ts infection and/or on day 15 after PbA infection.2.The survival of mice was monitored daily.The parasitemia of mice were monitored from day 3 after PbA infection.3.Collect the organs of mice,and conduct staining and testing.3.1 Mice were sacrificed at day 135 after Ts infection and/or at day 15 after PbA infection,each mouse body weight and liver weight were measured,and the liver index were calculated.3.2 Mice were sacrificed at day 135 after Ts infection and/or at day 15 after PbA infection,after H&E staining,liver pathology of mice was observed by light microscopy,and liver fibrosis of mice was observed by Sirius red staining.The F4/80+Kupffer cells and neutrophil in liver of mice were examined by immunohistochemical staining.3.3 After H&E staining,the pathologies of brain,lung,and small intestine of mice were observed by light microscopy at day 135 after Ts infection and/or at day 15 after PbA infection.3.4 Immunofluorescence staining for detecting NF-κB expression in the spleen tissue and positive areas were counted at day 135 after Ts infection and/or at day 15 after PbA infection.The mRNA expressions of TLRs[TLR1,TLR2,TLR3,TLR4,TLR5,TLR6,TLR7,TLR8,TLR9,TLR11,and TLR12]in spleen were detected by quantitative realtime reverse transcription-polymerase chain reaction(qRT-PCR).3.5 Mice were sacrificed at day 135 after Ts infection and/or at day 15 after PbA infection,the blood of mice were collected and centrifuged at 800 X g for 10 min,the supernatant was collected and stored at-20℃ until further use.The OD values of specific IgG,IgG1,IgG2a,and IgG2b which against PbA antigen,Ts larvae antigen,or PbA and Ts larvae mixed antigens were measured by ELISA detection method.Results1.Chronic T.spiralis infection cannot prolong the survival of mice co-infected with P.bergeri,but can reduce the peripheral red blood cell parasitemia.Chronic T.spiralis infection may reduce the thymus atrophy,spleen enlargement and attenuate pathological damage of liver,brain,and lungs,but may aggravate pathological damage of small intestine.2.Chronic T.spiralis infection may reduce liver pathology,inflammatory infiltration,and deposition of organ malaria pigment of mice co-infected with P.bergeri.Sirius red staining showed that positive area of liver fibrosis in Ts+PbA group was significantly higher than that in PbA group(P<0.05).Immunohistochemical staining showed that average optical density value of F4/80+Kupffer cells in Ts+PbA group was significantly higher than that in PbA group(P<0.01).3.Immunohistochemical staining showed that the average optical density value of NFκ B in spleen of Ts+PbA group was significantly lower than that in PbA group(P<0.05).qRT-PCR showed that,,the mRNA expression levels of TLR2,TLR3,and TLR6 in spleen of Ts+PbA group were significantly lower than that in PbA group(P<0.05).It is speculated that chronic T.spiralis infection of mice co-infected with P.bergeri may inhibit the expression of TLR2,TLR3,TLR6 and NF-κ B in the spleen,thereby exerting an immunomodulatory effect.4.The results showed that the OD values of specific IgG,IgG1,IgG2a,and IgG2b in PbA group and Ts+PbA group were significantly higher(P<0.05)than those of Ts group and naive group,which were against PbA and Ts larvae mixed antigens.There was no significant difference between PbA group and Ts+PbA group(P>0.05),which were against PbA antigen.Compared with Ts group,the OD values of specific IgG,IgG1,IgG2a,and IgG2b were significant increased in Ts+PbA group(P<0.05),which were against Ts larvae antigen.It suggests that chronic T.spiralis infection may increase the levels of antibodies those were against Ts larvae antigen in mice co-infected with P.bergeri.Conclusions1.Chronic T.spiralis infection may reduce the peripheral red blood cell parasitemia and attenuate pathological damage of liver,brain,and lung in mice co-infected with P.bergeri.2.Chronic T.spiralis infection may increase Kupffer cell expression and attenuate liver pathology in mice co-infected with P.bergeri.3.Chronic T.spiralis infection may inhibit the expression of TLR2,TLR3,TLR6,and NF-κ B in the spleen of mice co-infected with P.bergeri,thereby exerting an immunomodulatory effect.4.Chronic T.spiralis infection may increase the level of antibodies which against Ts larvae antigen in mice co-infected with P.bergeri.