thymopoietic and bone marrow response to murine ... · lineage commitment has been considered to...

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INFECTION AND IMMUNITY, May 2011, p. 2031–2042 Vol. 79, No. 5 0019-9567/11/$12.00 doi:10.1128/IAI.01213-10 Copyright © 2011, American Society for Microbiology. All Rights Reserved. Thymopoietic and Bone Marrow Response to Murine Pneumocystis Pneumonia Xin Shi, 1 Ping Zhang, 2 Gregory D. Sempowski, 3 and Judd E. Shellito 1 * Section of Pulmonary/Critical Care Medicine, Department of Medicine, 1 and Department of Physiology, 2 Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, and Duke Human Vaccine Institute, Duke University School of Medicine, Durham, North Carolina 27710 3 Received 15 November 2010/Returned for modification 13 December 2010/Accepted 4 February 2011 CD4 T cells play a key role in host defense against Pneumocystis infection. To define the role of naïve CD4 T cell production through the thymopoietic response in host defense against Pneumocystis infection, Pneumocystis murina infection in the lung was induced in adult male C57BL/6 mice with and without prior thymectomy. Pneumocystis infection caused a significant increase in the number of CCR9 multipotent progenitor (MPP) cells in the bone marrow and peripheral circulation, an increase in populations of earliest thymic progenitors (ETPs) and double negative (DN) thymocytes in the thymus, and recruitment of naïve and total CD4 T cells into the alveolar space. The level of murine signal joint T cell receptor excision circles (msjTRECs) in spleen CD4 cells was increased at 5 weeks post-Pneumocystis infection. In thymectomized mice, the numbers of naïve, central memory, and total CD4 T cells in all tissues examined were markedly reduced following Pneumocystis infection. This deficiency of naïve and central memory CD4 T cells was associated with delayed pulmonary clearance of Pneumocystis. Extracts of Pneumocystis resulted in an increase in the number of CCR9 MPPs in the cultured bone marrow cells. Stimulation of cultured bone marrow cells with ligands to Toll-like receptor 2 ([TLR-2] zymosan) and TLR-9 (ODN M362) each caused a similar increase in CCR9 MPP cells via activation of the Jun N-terminal protein kinase (JNK) pathway. These results demonstrate that enhanced production of naïve CD4 T lymphocytes through the thymopoietic response and enhanced delivery of lymphopoietic precursors from the bone marrow play an important role in host defense against Pneumocystis infection. Pneumocystis jirovecii is an opportunistic fungal pathogen causing severe pneumonia and pulmonary complications in immunocompromised hosts, particularly in individuals infected with the human immunodeficiency virus (HIV). CD4 T cells are known to play a key role in host defense against Pneumo- cystis infection (43). During HIV infection, activated memory CD4 T cells are the primary target cells destroyed by the virus (51). With the depletion of memory CD4 T cells, generation of naïve T lymphocytes from the thymus becomes a critical mechanism for the host to sustain enhanced T cell turnover (27, 42). Clinical investigations have shown that the thymic output of naïve CD4 T cells is activated in HIV-infected patients following highly active antiretroviral therapy (HAART) treatment (11, 38). This thymic activation is corre- lated with the increase in the number of naïve CD4 T cells and restoration of total CD4 T cell counts in the peripheral circulation (10, 16). Accumulated evidence suggests that the thymic output of naïve CD4 T cells may play an important role in maintaining or restoring immune function in immuno- compromised hosts. At this time, knowledge concerning the role of thymic production of naïve CD4 T cells in the host defense against Pneumocystis infection is lacking. Thymopoiesis requires continuous replenishment of lym- phoid progenitors from the bone marrow. In mouse bone mar- row, the most primitive hematopoietic stem cells (HSCs) with long-term (LT) self-renewal potential are enriched in the Lin c-Kit Sca-1 (LKS) cell fraction with the Thy1.1 lo (producing low levels of Thy1.1) or CD34 profile (2). These cells give rise to the short-term HSCs (ST-HSCs) or multipotent progenitors (MPPs) which are enriched in the Thy1.1 or CD34 LKS cell population. Lineage commitment has been considered to occur after the ST-HSC stage. Common lymphoid progenitors (CLPs) are among cells bearing the lLin c-Kit lo Sca-1 lo IL-7R (inter- leukin-7 receptor ) Thy1.1 phenotypic markers, which may serve as the precursors of both B and T cell lineages (20). A subset of marrow MPPs bearing CCR9 VCAM-1 (vascular adhesion molecule 1) surface markers has been identified as the lymphoid-specific precursors upstream of CLPs (24). Since CCR9 MPPs can more competitively home to the thymus than CLPs, they are considered the major marrow precursors of the earliest T lineage progenitors in the thymus (ETPs) (24). Upon homing to the thymus, the marrow precursors initially give rise to double negative (DN) thymocytes. These DN cells differentiate to express both CD4 and CD8 antigens, fol- lowed by acquisition of the CD3 antigen (double positive thymocytes). Passing through positive and negative selection processes, the matured T lymphocytes become single CD4 or CD8 cells. These matured single positive naïve T cells finally exit the thymus via efferent lymphatics. Thymic production of naïve T cells can be evaluated by the abundance of T cell receptor excision circles (TRECs) in pe- ripheral T cells (41). Murine signal joint TRECs (msjTRECs) are the episomal DNA circles generated during the rearrange- ment of the VDJ genes of the T cell receptor (TCR) and * Corresponding author. Mailing address: Section of Pulmonary/ Critical Care Medicine, LSU Health Sciences Center, 1901 Perdido Street, Room 3205, New Orleans, LA 70112. Phone: (504) 568-4634. Fax: (504) 568-4295. E-mail: [email protected]. Published ahead of print on 22 February 2011. 2031 on January 25, 2020 by guest http://iai.asm.org/ Downloaded from

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Page 1: Thymopoietic and Bone Marrow Response to Murine ... · Lineage commitment has been considered to occurT cells are the primary target cells destroyed by the virus (51). With the depletion

INFECTION AND IMMUNITY, May 2011, p. 2031–2042 Vol. 79, No. 50019-9567/11/$12.00 doi:10.1128/IAI.01213-10Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Thymopoietic and Bone Marrow Response toMurine Pneumocystis Pneumonia�

Xin Shi,1 Ping Zhang,2 Gregory D. Sempowski,3 and Judd E. Shellito1*Section of Pulmonary/Critical Care Medicine, Department of Medicine,1 and Department of Physiology,2

Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, andDuke Human Vaccine Institute, Duke University School of Medicine,

Durham, North Carolina 277103

Received 15 November 2010/Returned for modification 13 December 2010/Accepted 4 February 2011

CD4� T cells play a key role in host defense against Pneumocystis infection. To define the role of naïve CD4� Tcell production through the thymopoietic response in host defense against Pneumocystis infection, Pneumocystismurina infection in the lung was induced in adult male C57BL/6 mice with and without prior thymectomy.Pneumocystis infection caused a significant increase in the number of CCR9� multipotent progenitor (MPP) cellsin the bone marrow and peripheral circulation, an increase in populations of earliest thymic progenitors (ETPs) anddouble negative (DN) thymocytes in the thymus, and recruitment of naïve and total CD4� T cells into the alveolarspace. The level of murine signal joint T cell receptor excision circles (msjTRECs) in spleen CD4� cells wasincreased at 5 weeks post-Pneumocystis infection. In thymectomized mice, the numbers of naïve, central memory, andtotal CD4� T cells in all tissues examined were markedly reduced following Pneumocystis infection. This deficiencyof naïve and central memory CD4� T cells was associated with delayed pulmonary clearance of Pneumocystis.Extracts of Pneumocystis resulted in an increase in the number of CCR9� MPPs in the cultured bone marrow cells.Stimulation of cultured bone marrow cells with ligands to Toll-like receptor 2 ([TLR-2] zymosan) and TLR-9 (ODNM362) each caused a similar increase in CCR9� MPP cells via activation of the Jun N-terminal protein kinase(JNK) pathway. These results demonstrate that enhanced production of naïve CD4� T lymphocytes through thethymopoietic response and enhanced delivery of lymphopoietic precursors from the bone marrow play an importantrole in host defense against Pneumocystis infection.

Pneumocystis jirovecii is an opportunistic fungal pathogencausing severe pneumonia and pulmonary complications inimmunocompromised hosts, particularly in individuals infectedwith the human immunodeficiency virus (HIV). CD4� T cellsare known to play a key role in host defense against Pneumo-cystis infection (43). During HIV infection, activated memoryCD4� T cells are the primary target cells destroyed by the virus(51). With the depletion of memory CD4� T cells, generationof naïve T lymphocytes from the thymus becomes a criticalmechanism for the host to sustain enhanced T cell turnover(27, 42). Clinical investigations have shown that the thymicoutput of naïve CD4� T cells is activated in HIV-infectedpatients following highly active antiretroviral therapy(HAART) treatment (11, 38). This thymic activation is corre-lated with the increase in the number of naïve CD4� T cellsand restoration of total CD4� T cell counts in the peripheralcirculation (10, 16). Accumulated evidence suggests that thethymic output of naïve CD4� T cells may play an importantrole in maintaining or restoring immune function in immuno-compromised hosts. At this time, knowledge concerning therole of thymic production of naïve CD4� T cells in the hostdefense against Pneumocystis infection is lacking.

Thymopoiesis requires continuous replenishment of lym-phoid progenitors from the bone marrow. In mouse bone mar-

row, the most primitive hematopoietic stem cells (HSCs) withlong-term (LT) self-renewal potential are enriched in the Lin�

c-Kit� Sca-1� (LKS) cell fraction with the Thy1.1lo (producinglow levels of Thy1.1) or CD34� profile (2). These cells give riseto the short-term HSCs (ST-HSCs) or multipotent progenitors(MPPs) which are enriched in the Thy1.1� or CD34� LKS cellpopulation. Lineage commitment has been considered to occurafter the ST-HSC stage. Common lymphoid progenitors (CLPs)are among cells bearing the lLin� c-Kitlo Sca-1lo IL-7R�� (inter-leukin-7 receptor �) Thy1.1� phenotypic markers, which mayserve as the precursors of both B and T cell lineages (20). Asubset of marrow MPPs bearing CCR9� VCAM-1� (vascularadhesion molecule 1) surface markers has been identified as thelymphoid-specific precursors upstream of CLPs (24). SinceCCR9� MPPs can more competitively home to the thymus thanCLPs, they are considered the major marrow precursors of theearliest T lineage progenitors in the thymus (ETPs) (24).

Upon homing to the thymus, the marrow precursors initiallygive rise to double negative (DN) thymocytes. These DN cellsdifferentiate to express both CD4� and CD8� antigens, fol-lowed by acquisition of the CD3� antigen (double positivethymocytes). Passing through positive and negative selectionprocesses, the matured T lymphocytes become single CD4� orCD8� cells. These matured single positive naïve T cells finallyexit the thymus via efferent lymphatics.

Thymic production of naïve T cells can be evaluated by theabundance of T cell receptor excision circles (TRECs) in pe-ripheral T cells (41). Murine signal joint TRECs (msjTRECs)are the episomal DNA circles generated during the rearrange-ment of the VDJ genes of the T cell receptor (TCR) � and �

* Corresponding author. Mailing address: Section of Pulmonary/Critical Care Medicine, LSU Health Sciences Center, 1901 PerdidoStreet, Room 3205, New Orleans, LA 70112. Phone: (504) 568-4634.Fax: (504) 568-4295. E-mail: [email protected].

� Published ahead of print on 22 February 2011.

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chains in the thymus. These circles are stably retained duringcell division but do not replicate and therefore become dilutedamong daughter cells in peripheral lymphoid tissue.

To identify the significance of thymopoietic and bone mar-row activity in the host defense against Pneumocystis infection,we conducted experiments using both an in vivo model ofmurine Pneumocystis infection and in vitro cell cultures. Ourresults show that thymopoietic activity is enhanced followingintrapulmonary inoculation of Pneumocystis in adult mice. Thisthymopoietic response is supported by enhanced marrow gen-eration and delivery of thymopoietic precursor cells. Appro-priate bone marrow support and thymic output of naïve CD4�

T cells constitute important components of the host immunedefense against Pneumocystis infection.

MATERIALS AND METHODS

Animals. Specific-pathogen-free male C57BL/6 mice were purchased at 5weeks of age from NCI/Charles River Breeding Labs (Wilmington, MA). Ani-mals were housed in filter-topped cages and fed autoclaved chow and water adlibitum. All caging procedures and surgical manipulations were done under alaminar flow hood. These experimental protocols were performed in adherenceto the National Institutes of Health guidelines on the use of experimental ani-mals and with approval of the Institutional Animal Care and Use Committee atthe Louisiana State University Health Sciences Center.

Thymectomy. Thymectomy was performed using a previously described pro-cedure (37) which allows complete visualization of the entire thymus and itscomplete removal. Control mice received a sham operation.

Experimental design. Three weeks after thymectomy or sham operation, pul-monary infection with Pneumocystis was induced via intratracheal injection ofPneumocystis murina at a dose of 2 � 105 cysts per mouse (43). Animals weresacrificed at 1, 2, 3, 4, 5, and 6 weeks after the inoculation of Pneumocystis.Control mice were challenged with phosphate-buffered saline (PBS) alone.

Pneumocystis inoculation. Pneumocystis was prepared as described previouslyusing lung homogenates from chronically infected SCID mice (43). In brief,SCID mice chronically infected with Pneumocystis were injected with a lethaldose of pentobarbital. The animals were then exsanguinated by abdominal aortictransaction. The lungs were removed aseptically, placed in 1 ml of sterile PBS,and then frozen at �70°C. Frozen lungs were homogenized mechanically in 10ml of PBS by forcing tissue through a sterile 100-�m-pore-size nylon strainer(BD Biosciences, Bedford, MA) and centrifuged at 1,000 � g for 10 min at 4°C.The pellet was resuspended in PBS. Dilutions (1:5 and 1:10) of this suspensionwere stained with Giemsa stain (Diff-Quick; Dade Behring, Newark, DE). Thenumber of cysts was quantified microscopically, and the concentration of inoc-ulum was adjusted with PBS to 2 � 106 cysts/ml. Freshly prepared inoculum wasalways used for intratracheal inoculation to ensure viability of organisms. Re-cipient mice were anesthetized with intraperitoneal injection (i.p.) of ketamine/xylazine (200 mg/kg and 10 mg/kg, respectively). The trachea was surgicallyexposed. An 18-gauge blunt-ended needle was introduced into the tracheathrough the mouth under direct vision. Pneumocystis inoculum (2 � 105 Pneu-mocystis cysts in 0.1 ml) was injected through a 22-gauge inner needle into thelungs, followed by an injection of 0.3 ml of air to ensure adequate dispersion ofthe inoculum and clearance of the central airways. The neck incision was sutured,and the mice were placed prone for recovery.

Collection of cells from blood, bone marrow, thymus, lung-associated lymphnodes, and spleen. Upon sacrifice of animals, a heparinized blood sample wasobtained by cardiac puncture. After centrifugation at 500 � g for 10 min at roomtemperature, the plasma was collected and stored at �70°C for cytokine deter-mination. Femurs and tibias were collected, and bone marrow cells were flushedout with a total volume of 2 ml of PBS containing 2% bovine serum albumin(BSA; HyClone Laboratories, Logan, UT) through a 23-gauge needle. Bonemarrow cells were filtered through a 70-�m-pore-size nylon mesh (Sefar Amer-ica, Inc., Kansas City, MO). Tissue samples of the spleen, thymus, and lung-associated lymph nodes were collected and cut into small pieces (�3 mm). Thetissue pieces were placed in a Falcon cell strainer (70-�m-pore-size mesh size;BD Biosciences, Bedford, MA) set on top of a 50-ml centrifuge tube. Followinggentle pressure of tissue pieces against the strainer with a plunger from a 1-mlsyringe, the cells were filtered into the centrifuge tube by rinsing with 5 ml ofRPMI 1640 medium (ATCC, Manassas, VA). Contaminating red blood cells(RBCs) were lysed using RBC lysis solution (Gentra systems Minneapolis, MN).

After samples were washed with PBS, the recovered nucleated cells werecounted using a light microscope and hemacytometer.

CD4� splenocytes were isolated from single-cell suspensions of spleen cellsusing a MicroBeads protocol (Milteny Biotec, Auburn, CA). The purity ofisolated CD4� spleen cells was greater than 85% by flow cytometry.

Bronchoalveolar lavage (BAL). In a subset of animals, the trachea was exposedby a midline incision and cannulated with a polyethylene catheter. The lungswere lavaged with 10 ml of sterile Ca2�- and Mg2�-free PBS in 1-ml steps. Cellswere collected from the entire recovered BAL fluid by centrifugation at 300 � gfor 10 min at 4°C. Cell pellets were resuspended in PBS for counting in ahemacytometer and for flow cytometric analysis.

Flow cytometric analysis. Polychromatic (8 to 11 colors) phenotyping with flowcytometry was performed as described previously (56). Isolated blood leukocytesand nucleated bone marrow cells were suspended in RPMI 1640 medium con-taining 2% fetal calf serum (FCS) (2 � 106 cells in 100 �l of medium). The cellsuspension was added with a mixed panel of biotinylated anti-mouse lineagemarkers (10 �g/ml of each antibody against CD3e [clone 145-2C11], CD45R/B220 [clone RA3-6B2], CD11b/CD18 [Mac-1, clone M1/70), Gr-1 [Ly-6G/Ly-6C,clone RB6-8C5], and TER 119 [clone TER-119]) or isotype control antibodies(clones A 19-3, R35-95, and A95-1) (BD PharMingen, San Diego, CA). Follow-ing incubation for 20 min at 4°C, phycoerythrin (PE)-conjugated streptavidin (10�g/ml) and 10 �g/ml of each fluorochrome-conjugated anti-mouse CD117 (c-Kit,clone 2B8), anti-mouse Sca-1 (Ly-6A/E, clone D7), and anti-mouse CD34 (cloneRAM34), anti-mouse/rat CD90.1 (Thy1.1, clone HIS51), anti-mouse CD127(IL-7R�, clone A7R34), anti-mouse CCR9 (CDW199, clone CW-1.2), anti-mouse CD106 (VCAM-1, clone 429), and anti-mouse Ly-6A/E (Sca-1, clone D7)or the matched isotype control antibodies were added into the incubation system.The samples were further incubated in the dark for 20 min at 4°C. After cellswere washed with cold PBS, they were suspended in 0.5 ml of PBS containing 1%paraformaldehyde.

For analysis of thymocytes, BAL fluid cells, splenocytes, and lymphocytes oflung-associated lymph nodes, cells were suspended in RPMI 1640 medium con-taining 2% FCS (2 � 106 cells in 100 �l of medium) and fluorochrome-conju-gated antibodies (10 �g/ml of each) specific for murine CD3e (clone 145-2C11;BD Biosciences), CD4 (clone RM4-5; Invitrogen), CD8 (clone 53-6.7; eBio-Sciense), CD19 (1D3; BD Pharmingen), CD25 (P55, clone Pc61.5; eBioSciense),CD44 (Pgp-1, clone IM7; eBioSciense), CD62L (L-selectin, clone MEL-14;eBioSciense), F4-80 (BM8, clone BM8; eBioSciense), Ly-6G (Gr1, clone RB6-8c5; eBioSciense), CD117 (c-Kit, clone 2B8; eBioSciense), and Ly-6A/E (Sca-1,clone D7; eBioSciense) or isotype control antibodies. Following incubation for20 min at 4°C, the cells were washed with cold PBS and then fixed in 0.5 ml ofPBS containing 1% paraformaldehyde.

Analysis of cell phenotypes was performed on a FACSAria flow cytometerwith FACSDiva software (Becton Dickinson, San Jose, CA). Gating of each cellsubpopulation was set up with the reference of isotype-matched control antibodystaining in order to ensure precise determination of rare cell subtypes. In eachsample, 500,000 cells were acquired for analysis. Cell types designated by theirsurface makers are listed in Table 1.

RNA isolation and real-time RT-PCR for determination of PneumocystisrRNA. Upon sacrifice, the entire lung (right and left) of each mouse was re-moved. After the trachea, bronchi, and the surrounding connective tissue wereremoved, the whole lung was homogenized with TRIzol reagent to isolate totalRNA using protocols provided by the manufacturer (Invitrogen, Carlsbad, CA).To prepare an RNA standard for the assay, a portion of Pneumocystis murinarRNA (GenBank accession number AF257179) was cloned into PCR 2.1 Vector(Invitrogen, Carlsbad, CA), and Pneumocystis rRNA was produced by in vitrotranscription using T7 TNA polymerase (Promega, Madison, WI). TaqMan PCRprimers for mouse Pneumocystis rRNA were 5�-ATG AGG TGA AAA GTCGAA AGG G-3� and 5�-TGA TTG TCT CAG ATG AAA AAC CTC TT-3�. Theprobe was labeled with a reporter fluorescent dye, 6-carboxyfluorescein (FAM),and the sequence was 5�-6FAM-AACAGCCCAGAATAATGAATAAAGTTCCTCAATTGTTAC-TAMRA-3� (where TAMRA is 6-carboxytetramethylrhod-amine) (47). Real-time reverse transcription-PCR (RT-PCR) was performedusing a two-step method. Reverse transcription reactions were done in a volumeof 10 �l containing 200 ng of RNA sample, 1� TaqMan RT buffer, 5.5 mMmagnesium chloride, 500 �M each deoxynucleoside triphosphate (dNTP), 2.5�M random hexamer, 0.4 U/�l RNase inhibitor, and 1.25 U/�l MultiScribereverse transcriptase (Applied Biosystems, Branchburg, NJ). Samples were in-cubated at 25°C for 10 min and reverse transcribed at 48°C for 30 min; reversetranscriptase was inactivated at 95°C for 5 min. PCRs were done in a volume of50 �l containing 5 �l (100 ng) of cDNA, 1� TaqMan universal PCR Master Mix(Applied Biosystems, Branchburg, NJ), primers, and probe. An initial 2-minincubation was done at 50°C for uracyl N-glycosylase (UNG) activity to prevent

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a carryover reaction. The reaction was terminated by heating at 95°C for 5 min.The PCR amplification was performed for 40 cycles, with each cycle at 94°C for20 s and 60°C for 1 min. Data were converted to rRNA copy number using astandard curve of known copy Pneumocystis murina rRNA and are expressed ascopy number per lung.

Real-time PCR of msjTREC DNA expression. Murine signal joint T cellreceptor excision circles ([msjTRECs] GenBank AE008686) in CD4� spleencells were determined using the absolute quantitative real-time PCR protocoldeveloped by Gregory D. Sempowski’s laboratory at Duke University (41). Theresults are expressed as copies of msjTRECs/�g of DNA.

Determination of plasma mediators. Plasma levels of interleukin-3 (IL-3), IL-7,and IL-9 and of gamma interferon (IFN-) were determined by Luminex analysisusing a Milliplex MAP Kit (Millipore, Billerica, MA). The plasma level of Fms-related tyrosine kinase-3 (Flt-3) ligand was measured by enzyme-linked immunosor-bent assay (ELISA) using a murine Flt-3 ligand kit (R&D Systems, Minneapolis,MN).

Preparation of Pneumocystis extracts. Pneumocystis extracts were preparedusing a previously reported protocol (30) with some modifications. Sterility wasmaintained throughout the procedure of preparation. Pneumocystis-infectedmouse lungs were homogenized in ice-cold NKPC buffer (2.68 mM KCl, 1.47mM KH2PO4, 51.1 mM Na2HPO4, 7.43 mM NaH2PO4, 62 mM NaCl, 0.05 mMCaCl2, 0.05 mM MgCl2) containing 100 mM dithiothreitol. The homogenate wascentrifuged at 50 � g for 5 min at room temperature to remove cell debris.Pneumocystis cells in the supernatant were collected by centrifugation at10,000 � g for 10 min at 4°C. Pneumocystis cell pellets were then resuspended in5 ml of 0.85% NH4Cl-NKPC buffer and incubated at 37°C for 5 min to lyseerythrocytes. After samples were washed three times with cold NKPC buffer,isolated Pneumocystis organisms were quantified by enumeration of nucleistained with Giemsa stain. The host DNA was removed by incubating Pneumocystisextracts in 10 ml of NKPC buffer containing 0.02% (2 U) of DNase I type IV(Invitrogen, CA) at 37°C for 10 min. After samples were washed three times withcold NKPC buffer, isolated Pneumocystis organisms were suspended in NKPC bufferand subjected to ultrasonication for 20 s at 40 W and a 70% duty cycle (HeatSystems; Ultrasonics Inc., Plainview, NY) twice. Nuclei and cell ghosts were re-moved by centrifugation at 1,000 � g for 3 min. The supernatant representing theextract of 1 � 108 Pneumocystis cysts/ml was aliquoted and stored at �80°C.

In vitro culture of bone marrow cells. Nucleated bone marrow cells isolatedfrom naïve mice were plated into a 24-well tissue culture plate with 1 � 106 cellsper well in a total volume of 0.5 ml of StemSpan serum-free medium (StemCellTechnologies, Vancouver, BC, Canada). The cells were cultured at 37°C in anatmosphere of 5% CO2 for 16 h with 0.4 ml Pneumocystis extracts or ligands toTLR-2 (zymosan), TLR-4 (Escherichia coli lipopolysaccharide [LPS]), or TLR-9(ODN M362) (Invivogen, San Diego, CA) in the absence and presence ofspecific c-Jun kinase (JNK) inhibitor SP600125 (Sigma-Aldrich, St. Louis, MO).

Western blot analysis. A subset of cultured bone marrow cells was lysed witha lysis buffer (10 mM Tris-HCl, 1% Triton X-100, 5 mM EDTA, 50 mM NaCl,30 mM sodium pyrophosphate, 2 mM sodium orthovanadate [Na3VO4], 1 mMphenylmethylsulfonyl fluoride [PMSF], 50 mM sodium fluoride [NaF], 5 mg/mlaprotinin, 5 mg/ml pepstatin, and 5 mg/ml leupeptin, pH 7.6) to prepare celllysates. Western blot analysis of phospho-JNK levels in the cells was performedas described previously by our group (55). Protein concentrations of cell lysateswere determined using a bicinchoninic (BCA) protein assay kit (Pierce, Rock-ford, IL). Twenty micrograms of protein was resolved on 12% SDS-PAGE readygel (Bio-Red Laboratories, Hercules, CA) and then transferred onto a polyvi-nylidene difluoride (PVDF) membrane. The membrane was blocked with 5%fat-free milk in TSB-T buffer (10 mM Tris-HCl, 150 mM NaCl, 0.1% [vol/vol]Tween 20, 0.02% sodium azide, pH 7.4) and hybridized sequentially with primaryantibody against mouse phospho-JNK monoclonal IgG1 antibody (diluted1:1,000 with blocking buffer; Santa Cruz, CA) and horseradish peroxidase-con-jugated anti-mouse IgG (diluted 1:1,000 with blocking buffer; Cell Signaling,CA). Bound antibodies were detected by an ECL Plus Western blotting detec-tion kit (GE Healthcare, NJ). The blot was stripped with Re-Blot Plus mildantibody stripping solution (Millipore, Temecula, CA) following protocols sup-plied by the manufacturer. The membrane was reblotted with rabbit anti-�-actinantibody (Cell Signaling Technology, Danvers, MA) and horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling Technology, Danvers, MA) todetermine �-actin content in the sample loaded in each lane of the gel. Semi-quantification of the positive band in the images was performed using a KodakGel Logic 2,200 Imaging System. Data are presented as the net intensity ratio(NIR) of the phospho-JNK protein band versus the corresponding referenceprotein (�-actin) band.

Statistics. Data are presented as means standard errors of the means(SEM). The sample size is indicated in the legend to each figure. Statisticalanalysis was performed using GraphPad Prism, version 5, software (GraphPadSoftware, La Jolla, CA). Two-way analysis of variance and one-way analysis ofvariance followed by a Student-Newman Keuls test were used for comparisonsamong multiple groups. An unpaired Student’s t test was used for comparison oftwo different groups. Differences were considered statistically significant at a Pvalue of �0.05.

RESULTS

Changes in pulmonary Pneumocystis burden. To assess therole of the thymus in host defense against murine Pneumocystisinfection, clearance of Pneumocystis from the lung was exam-ined in mice without and with prior thymectomy. Healthy micehoused in filter-top cages have no detectable PneumocystisrRNA in whole-lung tissue. As shown in Fig. 1, after Pneumo-cystis infection, sham-operated mice showed an elevated levelof Pneumocystis rRNA in the lung at weeks 1 and 2 afterPneumocystis infection. Pneumocystis rRNA was then no lon-ger detectable by week 3 post-Pneumocystis inoculation, which

TABLE 1. Markers of cell typesa

Cell type Surface marker

LKS ............................................Lin� c-Kit� Sca-1�

HSC............................................Lin� c-Kit� Sca-1� Thy-1.1lo CD34�

MPP ...........................................Lin� c-Kit� Sca-1� Thy-1.1� CD34�

CCR9� MPP.............................Lin� c-Kit� Sca-1� Thy1.1� CD34�

CCR9� VCAM-1�

CLP ............................................Lin� c-Kitlo Sca-1lo Thy1.1� IL-7R� �

Lin� c-Kit� Sca-1� ..................Lin� c-Kit� Sca-1�

CMP...........................................Lin� c-Kit� Sca-1� IL-7R��

ETPb...........................................Lin (CD19 CD3 CD4 CD8 F4-80Gr-1)�

c-Kit� Sca-1� CD44� CD25�

DN1............................................CD4� CD8� CD44� CD25�

DN2............................................CD4� CD8� CD44� CD25�

DN3............................................CD4� CD8� CD44� CD25�

DN4............................................CD4� CD8� CD44� CD25�

T cellsCD4� .....................................CD3� CD4�

Naıve CD4� ..........................CD3� CD4� CD44lo CD62Lhi

Central memory CD4�........CD3� CD4� CD44hi CD62Lhi

Effect memory CD4�...........CD3� CD4� CD44hi CD62Llo

a See references 1-3, 13, 18-21, 23-26, and 34.b ETP, early thymic progenitor.

FIG. 1. Copies of Pneumocystis rRNA in the whole lung. *, P �0.05 versus week 1 sham-operated mice. Œ, P � 0.05 versus shamoperated mice at the same time point (n � 5 samples/week). W, week.

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indicates effective pulmonary clearance of the pathogen inthese animals. In contrast, mice with thymectomy showed aprogressive increase in Pneumocystis burden in the lung duringthe first 3 weeks of the infection. The level of PneumocystisrRNA in the lung then declined but remained elevated at week4 after inoculation. These results indicate that thymus activityplays an important but not essential role in host defenseagainst Pneumocystis infection in adult mice.

Changes in BAL fluid CD4� T cell subtypes. In response toPneumocystis inoculation, lymphocytes are recruited into lungtissue (47). Due to changes in thymic output of naïve CD4� Tcells and peripheral clonal expansion of memory T cells fol-lowing Pneumocystis infection, recruited CD4� T cell subpopu-lations in the alveolar space of animals without and withthymectomy may be altered accordingly. Therefore, we analyzedchanges in total numbers of CD4� T cells as well as CD4� T cellsubtypes in the alveolar space as reflected by cells recovered frombronchoalveolar lavage (BAL) fluid. As shown in Fig. 2, very fewCD4� T cells were recovered from BAL fluid of uninfected mice.At weeks 3 and 5 post-Pneumocystis infection, the proportions oftotal CD4� T cells as well as naïve, central memory, and effectormemory CD4� T cells in recovered BAL fluid cells were signifi-cantly increased in sham-operated mice. Thymectomy attenuatedthe increase in the numbers of total CD4� T cells, naïve CD4� Tcells, and effector memory CD4� T cells recovered by BAL atweeks 3 and 5 post-Pneumocystis inoculation. The fraction ofcentral memory CD4� T cells in BAL fluid at week 5 post-Pneumocystis infection was also lower in mice with thymectomythan in sham-operated animals.

Changes in CD4� T cell subtypes in peripheral lymphoidtissues and blood. In order to further understand the role ofthymic activity in the host defense response, we analyzedchanges in CD4� T cell populations as well as CD4� T cellsubtypes in the lung-associated lymph nodes, spleen, and sys-temic circulation.

In cells isolated from lung-associated lymph nodes, the pro-portions of total CD4� T cells, naïve CD4� T cells, and centralmemory CD4� T cells were persistently reduced in the 6-week-period post-Pneumocystis inoculation in sham-operated ani-mals (Fig. 3). Thymectomy caused an additional and significantdecrease in the naïve CD4� T cell subtype as well as totalCD4� T cells and central memory CD4� T cells in lung-associated lymph nodes compared to cell populations of thesham-operated controls. The effector memory CD4� T cellsubtype in lung-associated lymph nodes of the sham-operatedmice was initially reduced following Pneumocystis infection butrecovered by week 6 postinfection. Mice with thymectomyshowed the same level of effector memory CD4� T cells inlung-associated lymph nodes and a change in this subtype ofcells following Pneumocystis infection similar to that observedin sham-operated mice.

In the spleens of sham-operated mice, the fractions of totalCD4� T cells, naïve CD4� T cells, and central memory CD4�

T cells were initially reduced following Pneumocystis infection.These reductions were recovered at week 6 post-Pneumocystisinoculation (Fig. 3). Mice with thymectomy showed significantdecreases in the fraction of total CD4� T cells, naïve CD4� Tcells, and central memory CD4� T cells in the splenocytescompared to the sham-operated mice. These decreases werepersistent throughout the 6-week-period following Pneumocys-tis infection. In sham-operated mice, the number of effectormemory CD4� T cells in splenocytes was moderately reducedbetween 2 and 4 weeks of Pneumocystis infection but recoveredat week 5 following the infection. In mice with thymectomy, theproportion of effector memory CD4� T cells in splenocytes wasmaintained at a level similar to that of the sham-operatedmice. Pneumocystis infection was not associated with a reduc-tion of effector memory CD4� T cells in splenocytes of thymec-tomized mice.

The numbers of total CD4� T cells, naïve CD4� T cells, and

FIG. 2. CD4� cell types recovered from BAL fluid. * and ‡, P � 0.05 versus the corresponding control group (uninfected mice); Œ, P � 0.05versus sham-operated mice at the same time point (n � 5 samples/group).

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central memory CD4� T cells remained stable in the systemiccirculation of sham-operated mice following Pneumocystis in-fection (Table 2). The level of effector memory CD4� T cellsin the circulation was also stable during the initial 5 weeks afterPneumocystis inoculation in sham-operated mice. At week 6post-Pneumocystis inoculation, the circulating level of effectormemory CD4� T cells in sham-operated mice was significantlyelevated, suggesting that the influx of these effector cells intothe bloodstream exceeds their extravasation at this stage. Inmice with thymectomy, the number of total CD4� T cells in thesystemic circulation was markedly reduced compared to the

level in the sham operated animals. This reduction of totalCD4� cells in the circulation was persistent throughout the6-week-period post-Pneumocystis inoculation. The reductionof total CD4� T cells in the circulation of thymectomized miceprimarily resulted from the lack of naïve and central memoryCD4� T cells in the bloodstream (Table 2). In the meantime,the level of effector memory CD4� T cells in the circulationwas maintained in thymectomized mice compared to the levelin the sham-operated animals (Table 2).

Changes in ETP and DN cell populations in the thymus. Inorder to understand thymic functional activity following Pneu-

FIG. 3. CD4� cell types isolated from lung-associated lymph nodes (LN) (A) and spleen (B). * and ‡, P � 0.05 verus the corresponding controlgroup (uninfected mice); Œ, P � 0.05 versus Sham-operated mice at the same time point (n � 5 samples/group).

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mocystis infection, phenotypic analysis of thymocytes was per-formed using flow cytometry. As shown in Fig. 4A, the numberof ETPs in the thymus was significantly increased betweenweeks 1 and 4 following Pneumocystis infection. The number ofdouble negative stage 1 (DN1) cells in the thymus was signif-icantly increased in the first week of the infection (Fig. 4B).The number of double negative stage 2 (DN2) cells was in-creased throughout the 6-week-period of Pneumocystis infec-tion. Furthermore, the number of double negative stage 3(DN3) cells was also increased at weeks 1 and 2 of Pneumo-cystis infection. These results demonstrate that the pool oflymphoid precursor cells at different maturation stages is sig-nificantly expanded in the thymus following Pneumocystis in-fection. This expanded precursor cell pool in the thymus sup-ports enhanced thymic production of T lymphocytes.

Changes in msjTREC level in CD4� splenocytes. Measure-ment of msjTRECs in peripheral T cells has been used to studythymic output (41). The level of msjTRECs in CD4� spleno-cytes of sham-operated mice was decreased at week 1 post-Pneumocystis infection compared with the control level in un-infected mice (Fig. 4C). This initial decrease in msjTREC levelwas gradually attenuated between weeks 2 and 4 postinfection.At week 5 post-Pneumocystis inoculation, the msjTREC levelin CD4� splenocytes was markedly increased in sham-oper-ated mice, indicating enhanced homing of newly producednaïve CD4� lymphocytes to the spleen at this stage. In thymec-tomized animals, as expected, the level of msjTRECs in CD4�

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FIG. 4. The number of earliest thymic progenitors (ETPs)(A) and double negative (DN) cells (B) in the thymus.*, ‡, †, and ˆ,P � 0.05 versus the corresponding cell type in the control group(uninfected mice) (n � 5). (C) The level of msjTREC DNA inCD4� splenocytes. *, P � 0.05 verus the corresponding controlgroup (uninfected mice); Œ, P � 0.05 versus Sham-operated mice atthe same time point (n � 5 samples/group). D1-D4, DN1 to DN4cells (Table 1).

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splenocytes was significantly decreased throughout the 6-week-period following Pneumocystis inoculation.

Plasma levels of mediators. Plasma concentrations of IL-3and IL-7 were below detectable limits. Plasma levels of IL-9 andFlt-3 ligand were similar in sham-operated and thymectomizedmice and were not altered throughout the 6-week-period post-Pneumocystis infection in both groups (data not shown).

Changes in hematopoietic precursor populations in thebone marrow and blood. The enhancement of thymic outputrequires bone marrow support to provide thymopoietic pro-genitor cells. In order to understand the role of bone marrowduring this response, we examined the alteration of hemato-poietic precursor cell production in the bone marrow followingPneumocystis infection. As shown in Fig. 5A, the numbers ofmarrow LKS cells and HSCs increased following Pneumocystisinfection in mice without and with thymectomy. These in-creases in marrow LKS and HSC populations reached a peaklevel at weeks 2 and 3, respectively, post-Pneumocystis inocu-lation. Similarly, the number of marrow CCR9� MPPs (pre-cursors of ETPs) was significantly increased following Pneu-mocystis infection in both groups. The increase in marrowCCR9� MPPs reached a peak value at week 4 in sham-oper-ated animals and at week 5 in thymectomized mice. The bonemarrow pool of CLPs was slightly reduced in sham-operatedmice following Pneumocystis infection. In mice with thymec-tomy, the marrow CLP population was moderately increased atweeks 5 and 6 post-Pneumocystis infection. Bone marrow levelsof Lin� c-Kit� Sca-1� cells and CMPs were moderately re-duced in both the sham operation and thymectomy groups.

In association with the increase in the marrow pool of LKScells and CCR9� MPPs, the number of these precursors wassignificantly increased in the systemic circulation, with the peakvalues at weeks 1 and 3, respectively, post-Pneumocystis infec-tion in both groups (Fig. 5B). These results indicate that bonemarrow plays an important role in supporting the thymopoieticresponse to Pneumocystis infection. In contrast to what wasobserved in the bone marrow, the numbers of Lin� c-Kit�

Sca-1� cells and CMPs in the systemic circulation in sham-operated mice were increased during the initial 2 weeks post-Pneumocystis infection. Similarly, these two types of cells in thebloodstream were increased in thymectomized mice during theinitial 2 weeks and 1 week, respectively, following Pneumocystisinoculation.

Changes in hematopoietic precursor cell populations inbone marrow cells following in vitro culture with Pneumocystisextractions and TLR ligands. Bone marrow hematopoieticprecursor cells including primitive hematopoietic stem cellsexpress TLR receptors and respond to TLR ligand stimulation(14, 32, 52). In order to understand possible signaling mecha-nisms underlying the bone marrow precursor cell response toPneumocystis infection, we performed in vitro experiments inwhich bone marrow cells from naïve mice were cultured withPneumocystis extracts, the TLR-2 ligand zymosan, TLR-4 li-gand LPS, and TLR-9 ligand ODN M362. As shown in Fig. 6,the numbers of LKS cells, HSCs, MPPs, CCR9� MPPs, andCLPs were significantly increased in the cultured bone marrowcells following 16 h of exposure to Pneumocystis extracts and toligands of TLR-2, TLR-4, and TLR-9. In contrast, the numbersof Lin� c-Kit� Sca-1� cells and CMPs in the culture systemwere not increased (or even reduced) following exposure to

these stimulants. The dose-response test showed that zymosanand ODN M362 each caused a dose-dependent increase inLKS and CCR9� MPP cell types in cultured bone marrow cells(Fig. 7). These data demonstrate that TLR signaling may me-diate the bone marrow precursor cell response to Pneumocystisinfection.

JNK activation and the increase in CCR9� MPPs in bonemarrow cells. CCR9� MPPs are a subtype of LKS cells. Ourcurrent results showed that the numbers of LKS cells andCCR9� MPPs were increased in the bone marrow of mice withPneumocystis infection and in in vitro cultured marrow cellsfollowing exposure to Pneumocystis extracts and TLR ligands.In previous studies, we have observed that phenotypic conver-sion of Lin� c-Kit� Sca-1� cells by reexpression of Sca-1 is amajor mechanism underlying the rapid expansion of the mar-row LKS cell pool in response to infectious stimuli (22). Thepromoter region of the Sca-1 gene contains multiple bindingsites for AP1. c-Jun is the most potent transcriptional activatorin the AP1 family (31). Ligand engagement of TLR-2, TLR-4,or TLR-9 activates JNK, leading to enhancement of c-Juntranscriptional activity by phosphorylation of its N-terminalactivation domain (6, 14, 28, 31). Therefore, we examined therole of JNK signaling in Pneumocystis-mediated increases inLKS and CCR9� MPP cells. As shown in Fig. 8A, exposure ofcultured bone marrow cells to Pneumocystis extracts, zymosan,and ODN M362 for 8 h significantly increased JNK phosphor-ylation in these cells. Specific JNK inhibitor SP600125 pro-foundly inhibited the increase in the number of LKS cells aswell as LKS subtypes including CCR9� MPPs in cultured bonemarrow cells following exposure to Pneumocystis extracts, zy-mosan, and ODN M362 (Fig. 8B).

DISCUSSION

Thymic output of naïve T cells declines after puberty in bothhumans and mice (40, 48, 49). However, a thymic reserve maypersist into adulthood (36). In circumstances of accelerated Tlymphocyte turnover, such as those caused by chemotherapy orHIV disease, thymic function can be altered (5, 29, 35). Clin-ical investigations have shown that HIV-1-seropositive adultswith abundant thymic tissue exhibit both a higher percentageof naïve CD4� T cells and a higher total CD4� T cell count inthe circulation (29). Comparing HIV-2 and HIV-1 infectionshas revealed that HIV-2-infected patients demonstrate en-hanced thymic function compared to age-matched healthy in-dividuals (12). This activation of thymopoiesis is implicated inthe relative maintenance of CD4� T cell counts during HIV-2disease. Administration of growth hormone to HIV-1-infectedadults or interleukin-7 to simian immunodeficiency virus(SIV)-infected rhesus macaques under antiretroviral therapyincreases the thymic output of naïve T cells in these hosts (4,15, 33). It is well known that T cell replenishment in the bodyrelies on proliferation of existing T cells in the peripheraltissues and de novo naïve T cell production by the thymus. Ofthese two mechanisms, thymopoiesis is more efficient in restor-ing or replenishing the peripheral T cell profiles than clonalexpansion of existing peripheral T cells since peripheral expan-sion of T cells restricts the T cell repertoire to preexistingmemory T cells, which leads to inefficiency in responding tonew antigens (40).

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CD4� T cells are critical for the host defense against Pneu-mocystis infection (43, 44, 45, 46). Our previous studies haveshown that during Pneumocystis infection, the utilization ofperipheral CD4� T cells increases partially due to increased

destruction of these cells through enhanced apoptosis (47).This acceleration of CD4� T cell turnover requires increasedgeneration of CD4� T cells by the host. At this time, mecha-nisms underlying the replenishment of CD4� T cells remain

FIG. 5. Hematopoietic precursor cell types in the bone marrow (A) and circulation (B). * and ‡, P � 0.05 verus the corresponding control group(uninfected mice); Œ, P � 0.05 versus sham operated mice at the same time point (n � 5 samples/group). BMC, bone marrow cell; WBC, whiteblood cell.

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incompletely elucidated with regard to host defense againstPneumocystis infection. No information is available regardingthe significance of thymopoiesis in the host response to Pneu-mocystis infection.

The results of our current investigation show that blockingthymopoiesis in adult C57BL/6 mice by thymectomy delayedclearance of Pneumocystis from the lung following intratra-cheal inoculation. These data indicate that thymic functionconstitutes an important component of the host defenseagainst Pneumocystis infection. It has been known that HIV

infection and corticosteroid therapy both cause thymic toxicity.Thymic suppression in immunocompromised patients, partic-ularly those with AIDS or receiving long-term corticosteroidtherapy, may further impede the host defense against Pneu-mocystis infection. Our mice with thymectomy, however, wereeventually able to eradicate Pneumocystis infection in the lung,which suggests that peripheral clone expansion of existing Tcells in normal hosts can still sustain the host defense againstPneumocystis infection although in an inefficient manner. Ourcurrent study is in agreement with clinical observations in

FIG. 6. Changes in bone marrow cell types following culture with different stimuli. *, P � 0.05 versus the corresponding control group (n �5 samples/group). PC, Pneumocystis; ZYM, zymosan.

FIG. 7. Changes in the number of CCR9� MPPs and LKS cells in bone marrow cells following culture with different doses of TLR-2 and TLR-9ligands. *, P � 0.05 versus the corresponding control group (n � 5 samples/group).

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which no increase in incidence of Pneumocystis infection hasbeen reported in individuals previously thymectomized formyasthenia gravis (17).

In our murine model, immature thymocyte proliferation inthe thymus and thymic output of naïve CD4� T cells weresignificantly enhanced following Pneumocystis infection. Thenumbers of ETPs and DN thymocytes in the thymus of sham-operated mice were significantly increased after intratrachealinoculation of Pneumocystis. Similarly, the level of msjTRECsin CD4� splenocytes was significantly elevated in sham-oper-ated mice at 5 weeks post-Pneumocystis challenge. These re-sults support an activation of thymopoietic activity in adultmice in response to Pneumocystis infection. Interestingly, thelevel of msjTRECs in CD4� splenocytes was not increasedduring the initial 4 weeks post-Pneumocystis inoculation. Apossible explanation is that newly released naïve CD4� T cellsfrom the thymus may be preferentially recruited into the in-fected tissue site, i.e., the alveolar space. Our results demon-strated that the numbers of naïve CD4� T cells and total CD4�

T cells recruited into lung tissue were markedly increasedfollowing Pneumocystis infection. During this period of time,peripheral lymphoid tissues including the spleen and lung-associated lymph nodes primarily produce memory T cellsthrough enhanced clonal expansion. Increased homing of naïveCD4� T cells to these peripheral lymphoid tissues may con-tinue after clearance of the inoculated Pneumocystis from thelung. Mechanisms underlying this dynamic homing of naïveCD4� T cells during the host response to Pneumocystis infec-tion remain to be explored. Mice with thymectomy showed asignificant reduction of naïve CD4� T cell recruitment into the

lung following Pneumocystis infection, which suggests that pul-monary recruitment of these T cells was impaired in the ab-sence of appropriate thymopoietic support. In week 5 of Pneu-mocystis infection, the increase in central memory and effectormemory CD4� T cells in the lung was also attenuated in micewith thymectomy. It remains to be determined if this reductionof memory CD4� T cells in the lung resulted directly from aninsufficiency of naïve CD4� T cell conversion or involved othermechanisms yet to be identified.

Phenotypic analysis of CD4� T cells in the peripheral lym-phoid tissues and spleen shows a decrease in the numbers ofnaïve and central memory CD4� T cells but preservation ofeffector memory T cells following thymectomy. These altera-tions of CD4� T cell subtypes support the important role of thethymus in supporting naïve and central memory cells withinlymphoid tissue following a Pneumocystis challenge. These ob-servations also provide supporting evidence for the possibledynamic homing of these cells to an infected tissue site insteadof peripheral lymphoid tissues.

The active thymopoietic response to Pneumocystis infectionrequires thymopoietic progenitors supplied by the bone mar-row. Previous studies have shown that marrow CCR9� MPPsrepresent a major precursor population of ETPs (24). Ourcurrent results show that the marrow pool of CCR9� MPPswas expanded following Pneumocystis infection. This increasein the number of marrow CCR9� MPPs was accompanied bythe expansion of the upstream HSC pool as well as the entireLKS cell population in the bone marrow. With the increase inmarrow CCR9� MPPs, bone marrow release of these precur-sors into the systemic circulation was enhanced. As a conse-

FIG. 8. Phospho-JNK level in nucleated bone marrow cells following culture with TLR ligands (A). The image is a representative of four setsof cultures. *, P � 0.05 versus the control group. The effects of JNK inhibitor SP600125 (20 �M) on alteration of bone marrow cell types followingculture with different stimuli (B). *, P � 0.05 versus the control group; ‡, P � 0.05 versus cells cultured without SP600125 (n � 5 samples/group).

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quence, the number of CCR9� MPPs in the peripheral bloodwas significantly increased. In contrast to the increase in gen-eration of thymopoietic precursors, the numbers of Lin� c-Kit� Sca-1� cells and MPPs in the bone marrow were reduced,which suggests the polarization of marrow lineage supporttoward T cell production following Pneumocystis infection.These data identify the bone marrow as a key component ofthe thymopoietic response to Pneumocystis infection. In our invivo experiments, the levels of Lin� c-Kit� Sca-1� cells andMPPs were elevated in the bloodstream during the earlystage of Pneumocystis infection, which suggests an activatedmobilization of these cells into the systemic circulation. Thesignificance of this temporarily enhanced mobilization ofLin� c-Kit� Sca-1� cells and MPPs into the circulationfollowing Pneumocystis infection remains to be elucidated.One possible speculation for release of these precursorsfrom the bone marrow at the early stage of Pneumocystisinfection is that it may facilitate appropriate niche space forrapid expansion of the thymopoietic precursor cell popula-tion in the bone marrow.

Previous studies have demonstrated that bone marrow he-matopoietic precursor cells express TLRs, enabling these cellsto respond to TLR ligand stimulation (14, 32, 52). Pneumocys-tis is a fungal pathogen. Its cell components including glyco-protein and (1–3)-�-D-glucan can be recognized by TLRs, suchas TLR-2 and TLR-4 (9, 50, 52, 54). Prior investigations havenot addressed TLR-9 in Pneumocystis although this patternreceptor is important in recognition of other fungal pathogens(39). Clinical investigations and experimental studies have re-peatedly shown that circulating levels of Pneumocystis-derivedcell wall components are increased following pulmonary infec-tion with Pneumocystis (7, 8, 53). In preliminary studies, wealso observed an increase in plasma concentration of (1–3)-�-D-glucan in mice with Pneumocystis pneumonia (data notshown). Our current investigation shows that after in vitroexposure to Pneumocystis extracts, the numbers of LKS cells,HSCs, MPPs, CCR9� MPPs, and CLPs were significantly in-creased in cultured bone marrow cells. Culture of bone mar-row cells with the TLR-2 ligand zymosan, TLR-4 ligand LPS,and TLR-9 ligand ODN M362 resulted in a similar response.These data suggest that TLR signaling may be involved inmediating the alteration of the marrow hematopoietic precur-sor cell repertoire during Pneumocystis infection.

Recent studies from our group have revealed that marrowLin� c-Kit� Sca-1� cells can convert to LKS cells throughreexpression of Sca-1 in response to infectious stimuli (56).This phenotypic conversion of the downstream progenitors toupstream LKS cells serves as a major mechanism underlyingthe rapid expansion of the marrow LKS cell pool. In ourcurrent investigation, we also observed that in association withthe increase in marrow LKS cell and CCR9� MPP popula-tions, the number of Lin� c-Kit� Sca-1� cells in the bonemarrow was reduced in mice following Pneumocystis infection.Furthermore, culture of bone marrow cells with Pneumocystisextracts resulted in an increase in the number of LKS cells andCCR9� MPPs. Concomitantly, the number of Lin� c-Kit�

Sca-1� cells was reduced in the culture system. These datasupport the concept that upregulation of Sca-1 in marrowprecursor cells plays a critical role in reprogramming of theprecursor cell lineage commitment during the host response to

Pneumocystis infection. In studies previously reported by ourgroup, we have observed that mice with E. coli bacteremia alsoshow a rapid expansion of the marrow LKS cell population(56). Phenotypic conversion of Lin� c-Kit� Sca-1� cells toLKS cells by expression of Sca-1 plays a predominant role inthe expansion of the marrow LKS cell pool. This marrow LKScell response forms a platform for reprogramming the hema-topoietic precursor cell lineage commitment toward granulo-cyte production in the process of the host defense againstbacterial infection. These observations suggest that the hostdefense response to infectious pathogens may share a similarmechanism at the primitive hematopoietic precursor level.However, reprogramming of these precursor cells for selectedlineage commitment relies on unique signaling generated frominfection with a specific pathogen type.

By mapping the promoter region of the Sca-1 gene, weobserved that the Sca-1 promoter region contains multipleAP1 binding sites. c-Jun is the most potent transcriptionalactivator in the AP1 family (28). Studies have previously shownthat engagement of TLR-2, TLR-4, or TLR-9 with their li-gands activates JNK, leading to enhancement of c-Jun tran-scriptional activity by phosphorylation of its N-terminal acti-vation domain (14). Therefore, we determined if the JNKsignal pathway was involved in mediating the hematopoieticprecursor cell response to Pneumocystis infection. The resultsof our experiments showed that Pneumocystis extracts, zymo-san, and ODN M362 each activated JNK in cultured bonemarrow cells. Addition of the specific JNK inhibitor SP600125to the culture system profoundly inhibited the increase in LKScells and CCR9� MPPs in cultured bone marrow cells follow-ing exposure to Pneumocystis extracts, zymosan, or ODNM362. These findings demonstrate that the TLR-JNK-AP1signaling cascade may play a vital role in mediating the en-hancement of marrow T cell lineage support during the hostdefense response to Pneumocystis infection.

In our in vivo experiments, the plasma levels of IL-3, IL-7,IL-9, IFN-, and Flt-3 ligand were not altered. Previous inves-tigations have shown that these mediators may modulatethymopoietic activity. However, the role of these mediators (ifany) in our model of Pneumocystis infection remains undeter-mined.

In summary, thymopoietic activity is enhanced followingPneumocystis pneumonia in adult C57BL/6 mice along withincreases in thymopoietic precursor cells in the bone marrow.Ready access of Pneumocystis-derived TLR ligands into thesystemic circulation may stimulate bone marrow primitive he-matopoietic precursor cell reprogramming to enhance T lym-phocyte lineage commitment through activation of the JNK-AP1 pathway.

ACKNOWLEDGMENTS

We thank Joseph S. Sblosky, Connie P. Porretta, Jane A. Schexnay-der, and Amy B. Weinberg for their technical assistance.

This work was supported by National Institutes of Health grantsHL076100, AA017494, AA019676, and AG25150.

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