Graft-versus-host disease is the onset of an un- wanted immune response in patients who have undergone bone marrow transplantation [1]. The effect of this unwanted immune response is lethal in many cases. The goal of this research is to reduce the effect of graft-versus-host disease which can possibly reduce the waiting time for an eligible bone marrow donor to appear. This paper focused on the MyD88 adaptor protein, which triggers biochemical signals that can initiate, maintain, expand, or terminate inflammatory sites [2]. Inflammatory sites are the areas where the immune cells are gathered to fight foreign pathogens [1]. Experiments were carried out wherein stem cells were extracted from C57BL/6 mice and injected into BALB.B mice. This experiment design establishes the allogeneic bone marrow transplantation because the minor histocompatibility complex gene is un-matched between the two strains of mice while the major histocompatibility complex gene is matched. We focused on the effect of MyD88 protein deficiency in transplan- tation recipient. The positive/negative controls for the allogeneic bone marrow transplantation and MyD88 knockout allogeneic bone marrow transplantation group were carefully observed for 28 days and assessed for survival, weight, and immune cell fraction changes. We found that MyD88 protein deficiency yields prolonged survival in graft-versus-host disease. Data also suggests that innate immunity is the dominant factor in graft-versus- host disease, not adaptive immunity. Future experiments with cytokine analysis in a similar experiment design will provide more information about the relationship between the innate immunity and the severity of graft versus host disease

Choi, E. Y.

Choi, Sean

Jeon, J. Y.

Ju, J. M.

Lee, H. Y.

English

University of Calgary Journal Hosting

JOURNAL OF UNDERGRADUATE RESEARCH IN ALBERTA (JURA), VOL. 1, JULY 2011 19The Effect of MyD88 Deficiency DuringGraft-Versus-Host DiseaseSean Choi1, J. M. Ju2, J. Y. Jeon2, H. Y. Lee2, E. Y. Choi21Faculty of MedicineUniversity of Calgary3300 Hospital Drive NWCalgary, Alberta, Canada T2N 4N12Department of Biomedical SciencesSeoul National University College of MedicineSeoul, KoreaAbstract—Graft-versus-host disease is the onset of an un-wanted immune response in patients who have undergone bonemarrow transplantation [1]. The effect of this unwanted immuneresponse is lethal in many cases. The goal of this research is toreduce the effect of graft-versus-host disease which can possiblyreduce the waiting time for an eligible bone marrow donorto appear. This paper focused on the MyD88 adaptor protein,which triggers biochemical signals that can initiate, maintain,expand, or terminate inflammatory sites [2]. Inflammatory sitesare the areas where the immune cells are gathered to fight foreignpathogens [1]. Experiments were carried out wherein stem cellswere extracted from C57BL/6 mice and injected into BALB.Bmice. This experiment design establishes the allogeneic bonemarrow transplantation because the minor histocompatibilitycomplex gene is un-matched between the two strains of micewhile the major histocompatibility complex gene is matched. Wefocused on the effect of MyD88 protein deficiency in transplan-tation recipient. The positive/negative controls for the allogeneicbone marrow transplantation and MyD88 knockout allogeneicbone marrow transplantation group were carefully observed for28 days and assessed for survival, weight, and immune cellfraction changes. We found that MyD88 protein deficiency yieldsprolonged survival in graft-versus-host disease. Data also suggeststhat innate immunity is the dominant factor in graft-versus-host disease, not adaptive immunity. Future experiments withcytokine analysis in a similar experiment design will provide moreinformation about the relationship between the innate immunityand the severity of graft versus host disease.I. INTRODUCTIONHEMATOPOIETIC stem cell transplantation (HSCT),also commonly known as bone marrow transplantation,is a treatment option for hematological and immunologicaldefects, such as autoimmune disease and sickle-cell anaemia[3]. The general idea of HSCT is to firstly destroy thehematopoietic stem cells (HSC) as well as other immunecompetent cells in the patient to get rid of all defectivecells. Then replace them with a donors robust HSC. Thebenefit of performing HSCT is that you do not have to worryabout uncertainty whether treatment was effective or not.The disadvantage of HSCT is that it is an extremely riskyprocedure because of substantial side-effects. With currenttechnology advances, the surgery must be complimented withlifetime post-surgery-care to prevent lethal side-effects [3].In some cases, finding a completely MHC-matched donor isextremely difficult. In such cases, physicians can choose anon-ideal solution by performing allogeneic HSCT (usuallymatching at MHC locus but mis-matched at MiHC) along withsupplementary immunosuppressive treatments [4]. Ultimately,in order to achieve the most effective and safest allogeneicHSCT, the injected donor graft must be free of unwantedimmune cells. The major sources of unwanted immune cellare mature killer T cells. The recipients immune system willrespond and reject the donors mature killer T cells due topossibly the mismatched MHC or the minor histocompatibilityantigens (Minor H Ag) of the allogeneic immune competentcells [3].The unwanted onset of an immune response after HSCTis called graft-versus-host-disease (GVHD) [3]. It was widelyrecognized that the activation of the adaptive immune system isthe most influential factor of GVHD [1]. One possible way ofGVHD occurring is when the donors mature allogeneic T cellsare co-transported along with the HSC into the recipient [1].The mature T cells gather at the inflammatory site and attackthe recipients normal cells. Then, after enough time has passedfor the transplanted HSC to go through T cell development,those newly matured T cells will accelerate the damagingprocess. In order to do damage on the recipient cells, aninflammatory site is required. Without the inflammatory site,the damage done to the recipient will significantly decrease.It is worthwhile to investigate the causes of inflammationbecause prevention of inflammatory site formation will lead tono T cells being activated, and thus, no GVHD. Inflammatorysites can be formed via TLR signalling pathways.There are two major TLR signalling pathways: the MyD88-dependent pathway and the MyD88-independent pathway [1].TLR signalling pathways are needed to convert PAMPs intocommunicable nucleus signals [1]. PAMPs are found onpathogens, since they are usually an indispensable componentof the pathogens structure [2]. Some examples of PAMPsare lipopolysaccharides, double- and single-stranded RNA,glycoproteins, flagellin, profillin, and lipopeptides [1] Whenany of these PAMPs are recognized and bind to TLRs, asignal is transduced into the cell to mediate a response. In20 JOURNAL OF UNDERGRADUATE RESEARCH IN ALBERTA (JURA), VOL. 1, JULY 2011the MyD88-dependent pathway, the TLRs utilize the MyD88adapter protein to activate the transcription factor NF-?B. NF-?B then activates transcription, leading to the translatationof proteins necessary for the immune response. TLRs arelocated mainly on the cell membrane and endocytic vesicleof macrophages and dendritic cells [1].The hypothesis for this experiment was as follows: “defi-ciency of MyD88 protein in mice under GVHD condition willnot sufficiently form inflammatory environments, so that theinitiation of GVHD cannot take place in. Therefore, withoutthe inflammatory environment, activation of donor-originatedT cells would not be fully initiated. As a result, the donor graftsattack will be delayed because the cells have to find differentways to trigger inflammation. Therefore, the MyD88 deficientmouse will survive longer than the allogeneic control group.”This report has focused on the relationship between GVHDand MyD88-deficient mice to investigate on the relationshipbetween the GVHD and the innate immune system.II. METHOD & PROCEDUREA. Mice‘C57BL/6 strain HSC (spleen and bone marrow) intoBALB.B strain model’ was used because it is a well-established MHC-matched GVHD model (MiHC mis-matched) [2]. This will represent the allogeneic HSCT becauseC57BL/6 and BALB.B genetic strains are matched at MHC-loci to each other but most other background genes are mis-matched [2]. Donor and recipient mice were over 6 weeks oldand caged under specific pathogen-free environment. Over theduration of experiment, no more than 8 mice were kept in onecage to prevent psychological distress.B. Induction of GVHDThere were 3 experimental groups:i) One positive control (allogeneic HSCT) group: C57BL/6mouse strain into BALB.B mouse strain. The purpose ofthis group is to see the effect of allogeneic HSCT.ii) One negative control (Syngeneic HSCT) group: C57BL/6mouse strain into C57BL/6 mouse strain. The purpose ofthis group is to see the effect of lethal irradiation.iii) One experimental group: C57BL/6 mouse strain intoMyD88 knockout. BALB.B mouse strain. The purposeof this group is to see the effect of MyD88 knockoutmouse model in allogeneic HSCT.Recipient BALB.B mice were irradiated twice with 450 radwith a 5 hours interval. Then, the C57BL/6 donor mousesspleen and femur bone marrow cell graft were injected viaI.V. into the recipient BALB.B mouse. The preparation of thespleen and bone marrow cells are as follows:1) Spleen Cell Preparation: The extracted spleens werecollected in 1× PBS (8 g/L NaCl, 0.2 g/L KCl, 1.44 g/LNa2HPO4, 0.24 g/L KH2PO4 at 7.4 pH) solution and storedat 4 °C up to a maximum of 4 hours. In a PBS wetted 60mmdish, an autoclaved metal mesh was used to grind the spleen.Using a pipette, the spleen-PBS solution was transferred to a15 ml tube using cotton mesh for filtration. Then sample wascentrifuged (at 1500 rpm for 3 minutes) and the supernatantwas removed.2) Femur & Tibia Bone Marrow Preparation: The extractedfemur and tibia were collected in 1× PBS solution and storedat 4 °C up to a maximum of 4 hours. In a cold 100 mm dishon top of ice, a 3 ml syringe was used to extract bone marrowcells from femur and tibia. Using a pipette, the bone marrowsolution was transferred to a 15 ml tube using cotton meshfor filtration. The sample was centrifuged (at 1500 rpm for 3minutes) and the supernatant was removed.3) Donor Graft Preparation: The total volume of graftinjection was adjusted to 300µl per mouse using 1× PBS.The graft injection was calculated and adjusted so that eachmice received 2×107 spleen cells and 5×106 femur and tibiabone marrow cells.III. RESULTSThe Kaplan-Meier curve of mouse survival in Fig. 1 showsthat the allogeneic control group declined most rapidly amongthree experimental designs. At day 21, less than 20% of themice survived. The same graph also shows that the survivalrate of the MyD88 deficient group declined almost as slowlyas the syngeneic control group. At day 21, more than 70% ofthe mice survived. The Weight Change graph in Fig. 1 showsthat the MyD88 deficient group and allogeneic control grouphad similar weight change.Fig. 1. Kaplan-Meier curve of mouse survival (titled: GVHD Survival) andweight change of the 3 experimental groups over 28 days. Due to the fastinjection of the donor graft, a mouse in the allogeneic control group died onthe first day.Generally, graphs show that fractions rapidly increase untilday 4, then, either rapidly decreased or maintained its frac-tion except for the macrophage and the granulocytes. TheMacrophage Change graph and Granulocyte Change graphin Fig. 2 show that they continued to increase rapidly andreached the highest fraction (90%) by day 7. The macrophages,granulocytes, dendritic cells, B cells, and CD4+ T cells showeddistinct difference in positive and negative control groups.The Dendritic Cell Change graph in Fig. 3 shows an unusualfraction change. The dendritic cells in the MyD88 deficientgroup mice expectedly showed fraction change patterns assimilar to allogeneic control group until day 14. But after day14, the dendritic cell fraction notably increased and eventuallyreached at similar fraction as the syngeneic control group.THE EFFECT OF MYD88 DEFICIENCY DURING GRAFT-VERSUS-HOST DISEAS 21Fig. 2. Change in the fraction of innate immune cells. Each graph showsthe change that occurred in three different experimental groups over 28 daysusing FACS analysis. The result of 8 mice in each experimental group wascollected in to a solution together to acquire the mean value.Fig. 3. Change in the fraction of adaptive immune cells. Each graph showsany changes that occurred in three different experimental groups over 28 daysusing FACS analysis. The result of 8 mice in each experimental group wascollected in to a solution together to acquire the mean value.IV. DISCUSSIONThe hypothesis was supported, however the data show thereare more factors that need to be considered. For instance,the MyD88 knockout group lived longer than the allogeneiccontrol group. This pattern suggests that MyD88 deficient micemust hold mechanisms that keep them alive longer but still beseverely affected by GVHD.MyD88 is largely expressed in macrophages and dendriticcells [1]. It functions to send signals to transcription factorsin the nucleus to stimulate the production and secretion ofinflammatory signals [1]. Although most granulocytes do notnormally express MyD88 proteins, granulocytes do play awide range of roles during inflammation. Therefore it is logicalto see the macrophages and the granulocytes in allogeneicHSCT groups (Fig. 2) maintain very high fraction as shownin our results ??. We see that the high fraction of both B cellsand T cells demonstrate that the inflammatory site activityis much higher in the allogeneic HSCT models than in thesyngeneic models due to the GVHD effect.Macrophages have TLR receptor and MyD88 protein thatcan activate MyD88-dependent pathway. The main job ofmacrophages is to clean up after metabolic or immune ac-tivity via phagocytosis. Macrophages phagocytose pathogensand waste products after apoptosis or necrosis. Granulocytes(including all: neutrophil, basophil, eosinophils, mast cell, andnatural killer cells) act in many different roles in creatinginflammatory sites [1]. Noting the above two characteristicsof macrophages and granulocytes, we propose that the highfraction of macrophages and granulocytes show high incidenceof inflammation throughout the body. The fraction of theinnate immune cells which generate inflammatory sites inallogeneic HSCT group is about 45% higher than in syngeneicHSCT group, which strongly suggests that innate immunity isdominant in allogeneic HSCT during GVHD.The MyD88 deficient group shows a unique pattern in thedendritic cell fraction that is different than the allogeneicHSCT control group. The fraction of dendritic cell in theMyD88-deficient group is much higher in quantity than inthe allogeneic HSCT control group. But, we do not anticipatethat this pattern possesses any importance because the fractiondifferences between all three groups are very small anyways.However some counter-opinion may argue that as dendriticcells are known to act as messenger cells, it is realisticallypossible that the small quantity of dendritic cells can resultin significant consequences to the whole immune system.That is why the importance of this fraction pattern needsto be confirmed in future experiments. Once biochemistry ofthis pattern is explained in future experiments, more in-depthunderstanding of the relationship between the innate immunesystem and the adaptive immune system will be gained.B cells play an important role in adaptive immunity byproducing antibodies [5]. These antibodies are widely used forimmune attack via neutralization of epitopes, direct necrosis orsignalling indicators to other attacking cells [5]. The fractionof B cells in the syngeneic HSCT group is almost 5 foldgreater than the allogeneic HSCT group. This is the oppositeoutcome in comparison to expected outcome and warrantsinvestigation in future studies.The results described in this paper present important andstrong evidence to show that innate immune response ishighly active and a dominant factor in GVHD. However, thedesign of this experiment could not provide reasoning for thedendritic cell and B cell fraction patterns observed in GVHD.Future experiments should investigate the reason for the B cellfraction being very high in the syngeneic HSCT group but notin the allogeneic HSCT group. As well, the reason for MyD88deficient groups dendritic cells fraction pattern to change frombeing similar to allogeneic control group into being more-likesyngeneic control group.V. CONCLUSIONSOur data provided evidence to suggest that during GVHD,the number of the innate immune cells dominantly outnumbersthe adaptive immune cells. Therefore, it is more accurate tosay that the innate immune system is more important than the22 JOURNAL OF UNDERGRADUATE RESEARCH IN ALBERTA (JURA), VOL. 1, JULY 2011adaptive immune system during GVHD. Our results proposethat the innate immune strategies (inflammation without T-cell involvement) are most influential in GVHD. When theMyD88 protein is absent in GVHD, the adaptive immunesystem tries to compensate for the loss of its messengerfunction by producing more dendritic cells. But, we pre-sumed that increased dendritic cell is a very minor effectand has a minor contribution to the outcome of GVHD.On the contrary, the cells that show significant contributionto the outcome of GVHD are: macrophages, granulocytes(neutrophils, eosinophils, basophils, mast cell) and B cells.This report has identified and is suggesting several possibleresearch directions to study GVHD within MyD88 deficiency.No specific immune mechanism regarding MyD88 deficiencywas found in this report. The future experiments shouldbe focused on investigating cytokine secretion in blood ordifferent organs because the study of cytokines can tell usthe intensity and location of inflammation. The results foundin this experiment have offered a new perspective in thinkingabout GVHD; in particular, that it is possible to utilize theinnate immune system to suppress GVHD.REFERENCES[1] K. M. Murphy and et al., Janeway’s Immunobiology, 7th ed. Garland,2007.[2] C. A. Janeway and R. Medzhitov, “Innate immune recognition,” Immunol-ogy, Annual Reviews, vol. 20, pp. 197–216, 2002.[3] W. D. Shlomchik, “Graft-versus-host disease,” Immunology, Nature Re-view, vol. 7, pp. 340–352, 2007.[4] N. C. Institute. Bone marrow transplantation and peripheral blood stemcell transplantation.[5] B. Alberts, A. Johnson, and J. Lewis, Molecular Biology of the Cell,4th ed. New York: Garland Science, 2002, ch. Lymphocytes and theCellular Basis of Adaptive Immunity.

The Effect of MyD88 Deficiency During Graft-Versus-Host Disease

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The Effect of MyD88 Deficiency During Graft-Versus-Host Disease

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