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The Thymopoietic and Bone Marrow Response to Murine Pneumocystis Pneumonia 1

2

Xin Shi*, Ping Zhang, Gregory D. Sempowski

à, Judd E. Shellito*

§ 3

4

Section of Pulmonary/Critical Care Medicine, Department of Medicine*, and Department of 5

Physiology, LSU Health Sciences Center, New Orleans, LA 70112 6

Duke Human Vaccine Instituteà, Duke University School of Medicine, Durham, NC 27710 7

8

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Running title: The thymopoietic response to Pneumocystis pneumonia 10

11

§Correspondence: 12

Judd E. Shellito, MD 13

Section of Pulmonary/Critical Care Medicine 14

LSU Health Sciences Center 15

1901 Perdido Street, Room 3205 16

New Orleans, LA 70112 17

U.S.A. 18

Phone: (504) 568-4634 19

FAX: (504) 568-4295 20

Email: [email protected] 21

22

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.01213-10 IAI Accepts, published online ahead of print on 22 February 2011

on January 25, 2020 by guesthttp://iai.asm

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Abstract 23

CD4+ T cells play a key role in host defense against Pneumocystis infection. To define 24

the role of naïve CD4+ T cell production through the thymopoietic response in host defense 25

against Pneumocystis infection, Pneumocystis murina infection in the lung was induced in adult 26

male C57BL/6 mice with and without prior thymectomy. Pneumocystis infection caused a 27

significant increase in the number of CCR9+ multi-potent progenitor cells (CCR9

+MPPs) in the 28

bone marrow and peripheral circulation, an increase in populations of earliest thymic progenitors 29

(ETPs) and double negative (DN) thymocytes in the thymus, and recruitment of naïve and total 30

CD4+ T cells into the alveolar space. The level of murine single joint T cell receptor excision 31

circles (msjTRECs) in spleen CD4+ cells was increased at 5 weeks post Pneumocystis infection. 32

In thymectomized mice, the numbers of naïve, central memory, and total CD4+ T-cells in all 33

tissues examined were markedly reduced following Pneumocystis infection. This deficiency of 34

naïve and central memory CD4+ T cells was associated with delayed pulmonary clearance of 35

Pneumocystis. Extracts of Pneumocystis resulted in an increase in the number of CCR9+MPPs in 36

the cultured bone marrow cells. Stimulation of cultured bone marrow cells with ligands to TLR-2 37

(zymosan) and TLR-9 (ODN M362) each caused a similar increase in CCR9+MPPs via 38

activation of the JNK pathway. These results demonstrate that enhanced production of naïve 39

CD4+ T lymphocytes through the thymopoietic response and enhanced delivery of lymphopoietic 40

precursors from the bone marrow plays an important role in host defense against Pneumocystis 41

infection. 42


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Introduction 43

Pneumocystis jirovecii is an opportunistic fungal pathogen causing severe pneumonia and 44

pulmonary complications in immunocompromised hosts, particularly in individuals infected with 45

the human immunodeficiency virus (HIV). CD4+ T-cells are known to play a key role in host 46

defense against Pneumocystis infection (43). During HIV infection, activated memory CD4+ 47

T-cells are primary target cells destroyed by the virus (51). With the depletion of memory CD4+ 48

T-cells, generation of naïve T-lymphocytes from the thymus becomes a critical mechanism for 49

the host to sustain enhanced T cell turnover (27, 42). Clinical investigations have shown that the 50

thymic output of naïve CD4+ T-cells is activated in HIV-infected patients following highly active 51

antiretroviral therapy (HAART) treatment (11, 38). This thymic activation is correlated with the 52

increase in the number of naïve CD4+ T-cells and restoration of total CD4

+ T-cell counts in the 53

peripheral circulation (10, 16). Accumulated evidence suggests that the thymic output of naïve 54

CD4+ T-cells may play an important role in maintaining or restoring immune function in 55

immunocompromised hosts. At the present time, knowledge concerning the role of thymic 56

production of naïve CD4+ T-cells in host defense against Pneumocystis infection remains absent. 57

Thymopoiesis requires continuous replenishment of lymphoid progenitors from the bone 58

marrow. In mouse bone marrow, the most primitive hematopoietic stem cells (HSCs) with long-59

term (LT) self-renewal potential are enriched in the lin-c-kit

+Sca-1

+ (LKS) cell fraction with the 60

Thy1.1lo

or CD34- profile (2). These cells give rise to the short-term HSCs (ST-HSCs) or 61

multipotent progenitors (MPPs) which are enriched in the Thy1.1- or CD34

+ LKS cell population. 62

Lineage commitment has been considered to occur after the ST-HSC stage. Common lymphoid 63

progenitors (CLPs) are among cells bearing the lin-c-kit

lo Sca-1

lo IL-9Tí+

Thy1.1- phenotypic 64

markers, which may serve as the precursors of both B and T cell lineages (20). A subset of 65


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marrow MPPs bearing CCR9+VCAM-1

- surface markers has been identified as the lymphoid-66

specific precursors upstream of CLPs (24). Since CCR9+ MPPs can more competitively home to 67

the thymus compared with CLPs, they are considered the major marrow precursors of the earliest 68

T lineage progenitors in the thymus (ETPs) (24). 69

Upon homing to the thymus, the marrow precursors initially give rise to double negative 70

(DN) thymocytes. These DN cells differentiate to express both CD4+ and CD8

+ antigens 71

followed by acquisition of the CD3+ antigen (double positive thymocytes). Passing through 72

positive and negative selection processes, the matured T-lymphocytes become single CD4+ or 73

CD8+

cells. These matured single positive naïve T-cells finally exit the thymus via efferent 74

lymphatics. 75

Thymic production of naïve T-cells can be evaluated by the abundance of T cell receptor 76

excision circles (TRECs) in peripheral T cells (41). Murine signal joint TRECs are the episomal 77

DNA circles generated during the rearrangemepv"qh"vjg"XFL"igpgu"qh"vjg"VET"g"cpf"く chains in 78

the thymus. These circles are stably retained during cell division, but do not replicate, hence 79

becoming diluted among daughter cells in peripheral lymphoid tissue. 80

To identify the significance of thymopoietic and bone marrow activity in host defense 81

against Pneumocystis infection, we conducted experiments using both an in vivo model of 82

murine Pneumocystis infection and in vitro cell cultures. Our results show that thymopoietic 83

activity is enhanced following intrapulmonary inoculation of Pneumocystis in adult mice. This 84

thymopoietic response is supported by enhanced marrow generation and delivery of thymopoietc 85

precursor cells. Appropriate bone marrow support and thymic output of naïve CD4+ T-cells 86

constitutes an important component of host immune defense against Pneumocystis infection. 87

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Mater ials and Methods 89 90

Animals 91

Specific pathogen-free male C57BL/6 mice were purchased at 5 weeks of age from 92

NCI/Charles River Breeding Labs (Wilmington, MA). Animals were housed in filter-topped 93

cages and fed autoclaved chow and water ad libitum. All caging procedures and surgical 94

manipulations were done under a laminar flow hood. These experimental protocols were 95

performed in adherence to the National Institutes of Health guidelines on the use of experimental 96

animals and with approval of the Institutional Animal Care and Use Committee at the Louisiana 97

State University Health Sciences Center. 98

99

Thymectomy 100

Thymectomy was performed using a previously described procedure (37) which allows 101

complete visualization of the entire thymus and its complete removal. Control mice received a 102

sham operation. 103

104

Experimental Design 105

Three weeks after thymectomy or sham operation, pulmonary infection with 106

Pneumocystis was induced via intratracheal injection of Pneumocystis at a dose of 2 x 105 cysts 107

per mouse (43). Animals were sacrificed at 1, 2, 3, 4, 5 and 6 weeks after the inoculation of 108

Pneumocystis. Control mice were challenged with PBS alone. 109

110

Pneumocystis inoculation 111

Pneumocystis was prepared as described previously using lung homogenates from 112


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chronically infected SCID mice (43). In brief, SCID mice chronically infected with Pneumocystis 113

were injected with a lethal dose of pentobarbital. The animals were then exsanguinated by 114

abdominal aortic transaction. The lungs were removed aseptically, placed in 1 ml of sterile PBS 115

and then frozen at -70oC. Frozen lungs were homogenized mechanically in 10 ml of PBS by 116

forcing tissue through a sterile 100 µm nylon strainer (BD Biosciences, Bedford, MA) and 117

centrifuged at 1000 g for 10 min at 4oC. The pellet was resuspended in PBS. Dilutions (1:5 and 118

1:10) of this suspension were stained with Giemsa stain (Diff-Quick, Dade Behring, Newark, 119

DE). The number of cysts was quantified microscopically and the concentration of inoculum was 120

adjusted with PBS to 2 x 106 cysts/ml. Freshly prepared inoculum was always used for 121

intratracheal inoculation to ensure viability of organisms. Recipient mice were anesthetized with 122

intraperitoneal injection (IP) of ketamine/xylazine (200mg/kg and 10mg/kg respectively). The 123

trachea was surgically exposed. An 18-gauge blunt-ended needle was introduced into the trachea 124

through the mouth under direct vision. Pneumocystis inoculum (2 x 105 Pneumocystis cysts in 125

0.1 ml) was injected through a 22-gauge inner needle into the lungs followed by an injection of 126

0.3 ml of air to ensure adequate dispersion of the inoculum and clearance of the central airways. 127

The neck incision was sutured, and the mice were placed prone for recovery. 128

129

Collection of cells from blood, bone marrow, thymus, lung-associated lymph nods, and spleen 130

Upon sacrifice of animals, a heparinized blood sample was obtained by cardiac puncture. 131

After centrifugation at 500 x g for 10 min at room temperature, the plasma was collected and 132

stored at -70oC for cytokine determination. Femurs and tibias were collected and bone marrow 133

cells were flushed out with a total volume of 2 ml PBS containing 2% bovine serum albumin 134

(BSA, HyClone Laboratories, Logan, UT) through a 23-gauge needle. Bone marrow cells were 135


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filtered through a 70 micron nylon mesh (Sefar America INC. Kansas City, MO). Tissue samples 136

of the spleen, thymus, and lung-associated lymph nodes were collected and cut into small pieces 137

(~3 mm). The tissue pieces were placed in a Falcon cell strainer (70 µM mesh size, BD 138

Biosciences, Two Oak Park, Bedford, MA) set on top of a 50-ml centrifuge tube. Following 139

gentle pressure of tissue pieces against the strainer with a plunger from a 1 ml syringe, the cells 140

were filtered into the centrifuge tube via rinsing with 5 ml of RPMI-1640 medium (ATCC, 141

Manassas, VA). Contaminating red blood cells were lysed using RBC Lysis Solution (Gentra 142

systems Minneapolis, MN). After washing with PBS, the recovered nucleated cells were counted 143

using a light microscope and hemacytometer. 144

CD4+ splenocytes were isolated from single-cell suspensions of spleen cells using a 145

MicroBeads protocol (Milteny Biotec, Auburn, CA). The purity of isolated CD4+ spleen cells 146

was greater than 85% by flow cytometry. 147

148

Bronchoalveolar lavage (BAL) 149

In a subset of animals, the trachea was exposed by a midline incision and cannulated with 150

a polyethylene catheter. The lungs were lavaged with 10 ml of sterile Ca2+

and Mg2+

-free PBS in 151

1 ml steps. Cells were collected from the entire recovered BAL fluid by centrifugation at 300 g 152

for 10 min at 4oC. Cell pellets were resuspended in PBS for counting in a hemacytometer and for 153

flow cytometric analysis. 154

155

Flow cytometric analysis 156

Polychromatic (8 to 11 colors) phenotyping with flow cytometry was performed as 157

described previously (56). Isolated blood leukocytes and nucleated bone marrow cells were 158


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suspended in RPMI-1640 containing 2% fetal calf serum (FCS, 2 x 106 cells in 100 µl medium). 159

The cell suspension was added with a mixed panel of biotinylated anti-mouse lineage markers 160

[10 µg/ml of each antibody against CD3e (clone 145-2C11), CD45R/B220 (clone RA3-6B2), 161

CD11b/CD18 (Mac-1, clone M1/70), Gr-1 (Ly-6G/Ly-6C, clone RB6-8C5), and TER 119 (clone 162

TER-119)], or isotype control antibodies (clones A 19-3, R35-95, A95-1) (BD PharMingen, San 163

Diego, CA). Following incubation for 20 min at 4oC, PE-conjugated streptavidin (10 µg/ml) and 164

10 µg/ml of each fluorochrome conjugated anti-mouse CD117 (c-kit, clone 2B8), anti-mouse 165

Sca-1 (Ly-6A/E, clone D7), and anti-mouse CD34 (clone RAM34), anti-mouse/rat CD90.1 166

(Thy1.1, clone HIS51), anti-mouse CD127 (IL-9Tí. clone A7R34), anti-mouse CCR9 (CDW199, 167

clone CW-1.2), anti-mouse CD106 (VCAM-1, clone 429), and anti-mouse Ly-6A/E (Sca-1, 168

clone D7) or the matched isotype control antibodies were added into the incubation system. The 169

samples were further incubated in the dark for 20 min at 4oC. After washing with cold PBS, the 170

cells were suspended in 0.5 ml of PBS containing 1% paraformaldehyde. 171

For analysis of thymocytes, BAL cells, splenocytes, and lymphocytes of lung-associated 172

lymph nodes, the cells were suspended in RPMI-1640 containing 2% FCS (2 x 106 cells in 100 173

µl medium) and fluorochrome-conjugated antibodies (10 µg/ml of each) specific for murine 174

CD3e (clone 145-2C11, BD), CD4 (clone RM4-5, Invitrogen), CD8 (clone 53-6.7, eBioSciense), 175

CD19 (1D3, BD Pharmingen), CD25 (P55, clone Pc61.5, eBioSciense), CD44 (Pgp-1, clone IM7, 176

eBioSciense), CD62L (L-Selectin, clone MEL-14, eBioSciense), F4-80 (BM8, clone BM8, 177

eBioSciense), Ly-6G (Gr1, clone RB6-8c5, eBioSciense), CD117 (c-Kit, clone 2B8, 178

eBioSciense), and Ly-6A/E (Sca-1, clone D7, eBioSciense), or isotype control antibodies. 179

Following incubation for 20 min at 4oC, the cells were washed with cold PBS and then fixed in 180

0.5 ml of PBS containing 1% paraformaldehyde. 181


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Analysis of cell phenotypes was performed on a FACSAriaTM

flow cytometer with 182

FACSDiva software (Becton Dichinson, San Jose, CA). Gating of each cell subpopulation was 183

set up with the reference of isotype-matched control antibody staining in order to ensure precise 184

determination of rare cell subtypes. In each sample, 500,000 cells were acquired for analysis. 185

Cell types designated by their surface makers are listed in Table 1. 186

187

RNA isolation and real-time RT-PCR for determination of Pneumocystis rRNA 188

Upon sacrifice, the entire lung (right and left) of each mouse was removed. After 189

removing the trachea, bronchi, and the surrounding connective tissue, the whole lung was 190

homogenized with TRIzol reagent to isolate total RNA using protocols provided by the 191

manufacturer (Invitrogen, Carlsbad, CA). To prepare RNA standard for the assay, a portion of 192

Pneumocystis murina rRNA (GenBank Accession # AF257179) was cloned into PCR 2.1 Vector 193

(Invitrogen, Carlsbad, CA) and PC rRNA was produced by in vitro transcription using T7 TNA 194

polymerase (Promega, Madison, WI). TaqMan PCR primers for mouse Pneumocystis rRNA 195

were 5'- ATG AGG TGA AAA GTC GAAAGG G-3' and 5'-TGA TTG TCT CAG ATG AAA 196

AAC CTC TT-3'. The probe was labeled with a reporter fluorescent dye, 6-carboxyfluorescein 197

*HCO+."cpf"vjg"ugswgpeg"ycu"7Ó-198

6FAMAACAGCCCAGAATAATGAATAAAGTTCCTCAATTGTTACTAMRA-5Ó (47). Real-199

time RT-PCR was performed using a two step method. Reverse transcription reactions were done 200

in a volume of 10 µl containing 200ng RNA sample, 1xTaqMan RT buffer, 5.5 mM magnesium 201

ejnqtkfg."722たO"qh"gcej"fPVR."407たO"tcpfqo"jgzcogt."206"W1たn"Tpcug"kpjkdkvqt."3047"W1たn"202

MultiScribe reverse transcriptase, (Applied Biosystems, Branchbug, New Jersey). Samples were 203

incubated at 25ºC for 10 min, reverse transcribed at 48ºC for 30 min, reverse transcriptase 204


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inactivated at 95ºC for 5 min. PCR reactions were done in a vqnwog"qh"72"たn"eqpvckpkpi"7たn"205

(100ng) cDNA, 1x TaqMan universal PCR master mix (Applied Biosystems, Branchburg, New 206

Jersey), primers and probe. Initial 2 min incubation was done at 50ºC for UNG activity to 207

prevent carryover reaction. The reaction was terminated by heating at 95ºC for 5 min. The PCR 208

amplification was performed for 40 cycles with each cycle at 94ºC for 20 s and 60ºC for 1 min. 209

Data were converted to rRNA copy number using a standard curve of known copy Pneumocystis 210

murina rRNA and expressed as copy number per lung. 211

212

Real-time PCR of msjTREC DNA expression 213

Murine single joint T cell receptor excision circles (msjTRECs, GeneBank AE008686) in 214

CD4+ spleen cells were determined using the absolute quantitative real-time PCR protocol 215

developed by Dr. Gregory D. SempowskiÓu"ncdqtcvqt{"cv"Fwmg"University (41). The results are 216

expressed as copies of msjTRECs/µg DNA. 217

218

Determination of plasma mediators 219

Plasma levels of interleukin-3, 7, and 9 (IL-3, IL-7 and IL-9) and interferon-け"*KHP-220

け+ were determined by Luminex analysis using a MilliplexTM

MAP Kit (Millipore, Billerica, 221

MA). Plasma level of fms-related tyrosine kinase-3 (FLT3) ligand was measured by ELISA 222

using a murine FLT3 ligand kit (R&D systems, Minneapolis, MN). 223

224

Preparation of Pneumocystis extracts 225

Pneumocystis extracts were prepared using a previously reported protocol (30) with some 226

modifications. Sterility was maintained throughout the procedure of preparation. Pmeumocystis-227


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infected mouse lungs were homogenized in ice-cold NKPC buffer (2.68 mM KCL,1.47 mM 228

KH2PO4, 51.1 mM Na2HPO4, 7.43 mM NaH2PO4, 62 mM NaCL, 0.05 mM CaCL2, 0.05 mM 229

MgCL2) containing 100 mM dithiothreitol. The homogenate was centrifuged at 50 x g for 5 min 230

at room temperature to remove cell debris. Pneumocystis cells in the supernatant were collected 231

by centrifugation at 10,000 x g for 10 min at 4oC. Pneumocystis cell pellets were then 232

resuspended in 5 ml of 0.85% NH4CL-NKPC buffer and incubated at 37oC for 5 min to lyse 233

erythrocytes. After washing three times with cold NKPC buffer, isolated Pneumocystis 234

organisms were quantified by enumeration of nuclei stained with Giemsa stain. The host DNA 235

was removed by incubating Pneumocystis extracts in 10 ml of NKPC buffer containing 0.02% 236

(2U) DNase I type IV (invitrogene, CA) at 37o C for 10 min. After washing three times with cold 237

NKPC buffer, isolated Pneumocystis organisms were suspended in NKPC buffer and subjected 238

to ultrasonication for 20 second at 40 W and a 70% duty cycle (Heat Systems; Ultrasonics Inc., 239

Plainview, N.Y.) twice. Nuclei and cell ghosts were removed by centrifugation at 1,000 X g for 3 240

min. The supernatant representing the extract of 1 x 108 Pneumocystis cysts/ml was aliquoted 241

and stored at -80OC. 242

243

In vitro culture of bone marrow cells 244

Nucleated bone marrow cells isolated from naïve mice were plated into a 24-well tissue 245

culture plate with 1 x 106 cells per well in a total volume of 0.5 ml StemSpan serum-free medium 246

(StemCell Technologies, Vancouver, BC, Canada). The cells were cultured at 37oC in an 247

atmosphere of 5% CO2 for 16 h with 4 x 106

Pneumocystis extracts or ligands to TLR2 248

(Zymosan), TLR4 (E. coli LPS) or TLR9 (ODN M362) (Invivogen, SanDiego, CA) in the 249


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absence and presence of specific c-Jun kinase (JNK) inhibitor SP600125 (Sigma-Aldrich, St. 250

Louis, MO). 251

252

Western blot analysis 253

A subset of cultured bone marrow cells were lysed with a lysis buffer [10 mM Tris-HCl, 254

1% Triton X-100, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 2 mM sodium 255

orthovanadate (Na3VO4), 1 mM phenylmethylsulfonyl fluoride (PMSF), 50 mM sodium 256

fluoride (NaF), 5 mg/mL aprotinin, 5 mg/mL pepstatin, and 5 mg/mL leupeptin, pH 7.6] to 257

prepare cell lysates. Western blot analysis of phospho-JNK level in the cells was performed as 258

described previously by our group (55). Protein concentrations of cell lysates were determined 259

using a BCA protein assay kit (Pierce, Rockford, IL). Twenty micrograms of protein was 260

resolved on 12% SDS-PAGE ready gel (Bio-Red Laboratories, Hercules, CA) and then 261

transferred onto a PVDF membrane. The membrane was blocked with 5% fat free milk in TSB-T 262

buffer [10 mM Tris-HCl, 150 mM NaCl, 0.1% (vol/vol) Tween 20, 0.02% sodium azide, pH 7.4] 263

and hybridized sequentially with primary antibody against mouse phospho-JNK monoclonal 264

IgG1 antibody (1:1,000 with blocking buffer, Santa Cruz, CA) and horseradish peroxidase-265

conjugated anti-mouse IgG (1:1,000 with blocking buffer, Cell Signaling, CA) ). Bound 266

antibodies were detected by an ECL plus Western blotting detection kit (GE Healthcare, NJ). 267

The blot was stripped with Re-Blot plus mild antibody stripping solution (Millipore, Temecula, 268

CA) following protocols supplied by the manufacturer. The membrane was re-blotted with rabbit 269

anti-く-actin antibody (Cell Signaling Technology, Danvers, MA) and horseradish peroxidase-270

conjugated goat anti-rabbit IgG (Cell Signaling Technology, Danvers, MA) to determine く-actin 271

content in the sample loaded in each lane of the gel. Semi-quantification of the positive band in 272


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the images was performed using a Kodak Gel Logic 2,200 Imaging System. Data are presented 273

as the net intensity ratio (NIR) of the phospho-JNK protein band versus the corresponding 274

reference protein (く-actin) band. 275

276

Statistics 277

Data are presented as mean ± SEM. The sample size is indicated in the legend to each 278

figure. Statistical analysis was performed using GraphPad Prism 5 software (GraphPad Software, 279

La Jolla, CA). Two-way analysis of variance and one-way analysis of variance followed by the 280

Student-Newman Keuls test were used for comparisons among multiple groups. Unpaired 281

UvwfgpvÓu"t-test was used for comparison of 2 different groups. Differences were considered 282

statistically significant at p < 0.05. 283

284


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Results 285

Changes in pulmonary Pneumocystis burden 286

To assess the role of the thymus in host defense against murine Pneumocystis infection, 287

clearance of Pneumocystis from the lung was examined in mice without and with prior 288

thymectomy. Normal mice housed in filter top cages have no detectable Pneumocystis rRNA in 289

whole lung tissue. As shown in Figure 1, after Pneumocystis infection, sham operated mice 290

showed an elevated level of Pneumocystis rRNA in the lung at weeks 1 and 2 after Pneumocystis 291

infection. Pneumocystis rRNA was then no longer detectable by week 3 post Pneumocystis 292

inoculation, which indicates effective pulmonary clearance of the pathogen in these animals. In 293

contrast, mice with thymectomy showed a progressive increase in Pneumocystis burden in the 294

lung during the first 3 weeks of the infection. The level of Pneumocystis rRNA in the lung then 295

declined but remained elevated at week 4 after inoculation. These results indicate that thymus 296

activity plays an important but not essential role in host defense against Pneumocystis infection 297

in adult mice. 298

299

Changes in bronchoalveolar lavage (BAL) CD4+ T-cell subtypes 300

In response to Pneumocystis inoculation, lymphocytes are recruited into lung tissue (47). 301

Due to changes in thymic output of naïve CD4+ T-cells and peripheral clonal expansion of 302

memory T-cells following Pneumocystis infection, recruited CD4+ T-cell subpopulations in the 303

alveolar space of animals without and with thymectomy may be altered accordingly. Therefore, 304

we analyzed changes in total numbers of CD4+ T cells as well as CD4

+ T-cell subtypes in the 305

alveolar space as reflected by cells recovered from BAL fluid. As shown in Figure 2, very few 306

CD4+ T-cells were recovered from BAL fluid of uninfected mice. At weeks 3 and 5 post 307


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Pneumocystis infection, the proportions of total CD4+ T-cells as well as naïve, central memory, 308

and effector memory CD4+ T-cells in recovered BAL cells were significantly increased in sham 309

operated mice. Thymectomy attenuated the increase in the numbers of total CD4+ T-cells, naïve 310

CD4+ T-cells, and effector memory CD4

+ T-cells recovered by BAL at weeks 3 and 5 post 311

Pneumocystis inoculation. The fraction of central memory CD4+ T-cells in BAL at week 5 post 312

Pneumocystis infection was also lower in mice with thymectomy than in sham operated animals. 313

314

Changes in CD4+ T-cell subtypes in peripheral lymphoid tissues and blood 315

In order to further understand the role of thymic activity in the host defense response, we 316

analyzed changes in CD4+ T cell populations as well as CD4

+ T-cell subtypes in the lung-317

associated lymph nodes, spleen, and systemic circulation. 318

In cells isolated from lung associated lymph nodes, the proportions of total CD4+ T-cells, 319

naïve CD4+ T-cells, and central memory CD4

+ T-cells were persistently reduced in the 6 week 320

period post Pneumocystis inoculation in sham operated animals (Figure 3). Thymectomy caused 321

an additional and significant decrease in the naïve CD4+

T-cell subtype as well as total CD4+ T-322

cells, and central memory CD4+ T-cells in lung-associated lymph nodes compared to the sham 323

operated controls. The effector memory CD4+ T-cell subtype in lung associated lymph nodes of 324

the sham operated mice was initially reduced following Pneumocystis infection, but recovered by 325

week 6 post the infection. Mice with thymectomy showed the same level of effector memory 326

CD4+ T-cells in lung associated lymph nodes and a similar change in this subtype of cells 327

following Pneumocystis infection as observed in sham operated mice. 328

In the spleens of sham operated mice, the fractions of total CD4+ T-cells, naïve CD4

+ T-329

cells, and central memory CD4+ T-cells were initially reduced following Pneumocystis infection. 330


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These reductions were recovered at week 6 post Pneumocystis inoculation (Figure 3). Mice with 331

thymectomy showed significant decreases in the fraction of total CD4+ T-cells, naïve CD4

+ T-332

cells, and central memory CD4+ T-cells in the splenocytes as compared to the sham operated 333

mice. These decreases were persistent throughout the 6 week period following Pneumocystis 334

infection. In sham operated mice, the number of effector memory CD4+ T-cells in splenocytes 335

was moderately reduced between 2 to 4 weeks of Pneumocystis infection, but recovered at week 336

5 following the infection. In mice with thymectomy, the proportion of effector memory CD4+ T-337

cells in splenocytes was maintained at a level similar to the sham operated mice. Pneumocystis 338

infection was not associated with a reduction of effector memory CD4+ T-cells in splenocytes of 339

thymectomized mice. 340

The numbers of total CD4+ T-cells, naïve CD4

+ T-cells, and central memory CD4

+ T-341

cells remained stable in the systemic circulation of sham operated mice following Pneumocystis 342

infection (Table 2). The level of effector memory CD4+ T-cells in the circulation was also stable 343

during the initial 5 weeks after Pneumocystis inoculation in sham operated mice. At week 6 post 344

Pneumocystis inoculation, the circulating level of effector memory CD4+ T-cells in sham 345

operated mice was significantly elevated, suggesting the influx of these effector cells into the 346

blood stream exceeds their extravasation at this stage. In mice with thymectomy, the number of 347

total CD4+ T-cells in the systemic circulation was markedly reduced as compared to the sham 348

operated animals. This reduction of total CD4+ cells in the circulation was persistent throughout 349

the 6 week period post Pneumocystis inoculation. The reduction of total CD4+ T-cells in the 350

circulation of thymectomized mice primarily resulted from the lack of naïve and central memory 351

CD4+ T-cells in the blood stream (Table 2). In the meantime, the level of effector memory CD4

+ 352


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T-cells in the circulation was maintained in thymectomized mice as compared to the level in the 353

sham operated animals (Table 2). 354

355

Changes in ETP and DN cell populations in the thymus 356

In order to understand thymic functional activity following Pneumocystis infection, 357

phenotypic analysis of thymocytes was performed using flow cytometry. As shown in Figure 4A, 358

the number of ETPs in the thymus was significantly increased between weeks 1 and 4 following 359

Pneumocystis infection. The number of double negative stage 1 (DN1) cells in the thymus was 360

significantly increased in the 1st week of the infection (Figure 4B). The number of double 361

negative stage 2 (DN2) cells was increased throughout the 6 week period of Pneumocystis 362

infection. Furthermore, the number of double negative stage 3 (DN3) cells was also increased at 363

weeks 1 and 2 of Pneumocystis infection. These results demonstrate that the pool of lymphoid 364

precursor cells at different maturation stages is significantly expanded in the thymus following 365

Pneumocystis infection. This expanded precursor cell pool in the thymus supports enhanced 366

thymic production of T-lymphocytes. 367

368

Changes in msjTREC level in CD4+ splenocytes 369

Measurement of msjTRECs in peripheral T cells has been used to study thymic output 370

(41). The level of msjTRECs in CD4+ splenocytes of sham-operated mice was decreased at week 371

1 post Pneumocystis infection as compared with the control level in uninfected mice (Figure 4C). 372

This initial decrease in msjTRECs level was gradually attenuated between weeks 2 to 4 post 373

infection. At week 5 post Pneumocystis inoculation, msjTRECs level in CD4+ splenocytes was 374

markedly increased in sham operated mice indicating enhanced homing of newly produced naïve 375


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CD4+ lymphocytes to the spleen at this stage. In thymectomized animals, as expected, the level 376

of msjTRECs in CD4+ splenocytes was significantly decreased throughout the 6 week period 377

following Pneumocystis inoculation. 378

379

Plasma levels of mediators 380

Plasma concentrations of IL-3 and IL-7 were below detectable limits. Plasma levels of 381

IL-9 and Flt-3 ligand were similar in sham operated and thymectomized mice and were not 382

altered throughout the 6 week period post Pneumocystis infection in both groups (data not 383

shown). 384

385

Changes in hematopoietic precursor populations in the bone marrow and blood 386

The enhancement of thymic output requires bone marrow support to provide 387

thymopoietic progenitor cells. In order to understand the role of bone marrow during this 388

response, we examined the alteration of hematopoietic precursor cell production in the bone 389

marrow following Pneumocystis infection. As shown in Figure 5A, the numbers of marrow LKS 390

cells and HSCs increased following Pneumocystis infection in mice without and with 391

thymectomy. These increases in marrow LKS and HSC populations reached a peak level at 392

weeks 2 and 3, respectively, post Pneumocystis inoculation. Similarly, the number of marrow 393

CCR9+MPPs (precursors of ETPs) was significantly increased following Pneumocystis infection 394

in both groups. The increase in marrow CCR9+MPPs reached a peak value at week 4 in sham 395

operated animals and at week 5 in thymectomized mice. The bone marrow pool of CLPs was 396

slightly reduced in sham operated mice following Pneumocystis infection. In mice with 397

thymectomy, the marrow CLP population was moderately increased at weeks 5 and 6 post 398


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Pneumocystis infection. Bone marrow levels of lin-c-kit

+Sca-1

- cells and CMPs were moderately 399

reduced in both sham operation and thymectomy groups. 400

In association with the increase in the marrow pool of LKS cells and CCR9+MPPs, the 401

number of these precursors was significantly increased in the systemic circulation with the peak 402

values at weeks 1 and 3, respectively, post Pneumocystis infection in both groups (Figure 5B). 403

These results indicate that bone marrow plays an important role in supporting the thymopoietic 404

response to Pneumocystis infection. In contrast to what was observed in the bone marrow, the 405

numbers of lin-c-Kit

+Sca-1

- cells and CMPs in the systemic circulation in sham operated mice 406

were increased during the initial 2 weeks post Pneumocystis infection. Similarly, these two types 407

of cells in the blood stream were increased in thymectomized mice during the initial 2 and 1 408

week, respectively, following Pneumocystis inoculation. 409

410

Changes in hematopoietic precursor cell populations in bone marrow cells following in vitro 411

culture with Pneumocystis extractions and TLR ligands 412

Bone marrow hematopoietic precursor cells including primitive hematopoietic stem cells 413

express TLR receptors and respond to TLR ligand stimulation (14, 32, 52). In order to 414

understand possible signaling mechanisms underlying the bone marrow precursor cell response 415

to Pneumocystis infection, we performed in vitro experiments in which bone marrow cells from 416

naïve mice were cultured with Pneumocystis extracts, TLR-2 ligand Zymosan, TLR-4 ligand 417

LPS, and TLR-9 ligand ODN M362. As shown in Figure 6, the numbers of LKS cells, HSCs, 418

MPPs, CCR9+MPPs, and CLPs were significantly increased in the cultured bone marrow cells 419

following 16 h exposure to Pneumocystis extracts and ligands to TLR-2, TLR-4, and TLR-9. In 420

contrast, the numbers of lin-c-kit

+Sca-1

- cells and CMPs in the culture system were not increased 421


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(or even reduced) following exposure to these stimulants. The dose response test showed that 422

Zymosan and ODN M362 each caused a dose-dependent increase in LKS and CCR9+MPP cell 423

types in cultured bone marrow cells (Figure 7). These data demonstrate that TLR signaling may 424

mediate the bone marrow precursor cell response to Pneumocystis infection. 425

426

JNK activation and the increase in CCR9+MPPs in bone marrow cells 427

CCR9+MPPs are a subtype of LKS cells. Our current results showed that the numbers of 428

LKS cells and CCR9+MPPs were increased in the bone marrow of mice with Pneumocystis 429

infection and in in vitro cultured marrow cells following exposure to Pneumocystis extracts and 430

TLR ligands. In previous studies, we have observed that phenotypic conversion of lin-c-Kit

+Sca-431

1- cells by re-expression of Sca-1 is a major mechanism underlying the rapid expansion of 432

marrow LKS cell pool in response to infectious stimuli (22). The promoter region of the Sca-1 433

gene contains multiple binding sites for AP1. C-Jun is the most potent transcriptional activator in 434

the AP1 family (31). Ligand engagement of TLR-2, TLR-4, and TLR-9 all activate JNK leading 435

to enhancement of c-Jun transcriptional activity by phosphorylation of its N-terminal activation 436

domain (6, 14, 28, 31). Therefore, we examined the role of JNK signaling in Pneumocystis-437

mediated increases in LKS and CCR9+MPP cells. As shown in Figure 8A, exposure of cultured 438

bone marrow cells to Pneumocystis extracts, Zymosan, and ODN M362 for 8 hours significantly 439

increased JNK phosphorylation in these cells. Specific JNK inhibitor SP600125 profoundly 440

inhibited the increase in the number of LKS cells as well as LKS subtypes including 441

CCR9+MPPs in cultured bone marrow cells following exposure to Pneumocystis extracts, 442

Zymsan, and ODN M362 (Figure 8B). 443

444


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Discussion 445

Thymic output of naïve T-cells declines after puberty in both humans and mice (40, 48, 446

49). However, a thymic reserve may persist into adulthood (36). In circumstances of accelerated 447

T-lymphocyte turnover such as those caused by chemotherapy or HIV disease, thymic function 448

can be altered (5, 29, 35). Clinical investigations have shown that HIV-1-seropositive adults with 449

abundant thymic tissue exhibit both a higher percent of naïve CD4+ T-cells and a higher total 450

CD4+ T-cell count in the circulation (29). Comparing HIV-2 versus HIV-1 infections has 451

revealed that HIV-2-infected patients demonstrate enhanced thymic function compared to the 452

age-matched healthy individuals (12). This activation of thymopoiesis is implicated in the 453

relative maintenance of CD4+ T-cell counts during HIV-2 disease. Administration of growth 454

hormone to HIV-1infected adults or interleukin-7 to SIV-infected rhesus macaques under 455

antiretroviral therapy increases the thymic output of naïve T-cells in these hosts (4, 15, 33). It is 456

well known that T-cell replenishment in the body relies on proliferation of existing T-cells in the 457

peripheral tissues and de novo naïve T-cell production by the thymus. Of these two mechanisms, 458

thymopoiesis is more efficient in restoring or replenishing the peripheral T-cell profiles than is 459

clonal expansion of existing peripheral T-cells, since peripheral expansion of T-cells restricts the 460

T-cell repertoire to preexisting memory T-cells which leads to inefficiency in responding to new 461

antigens (40). 462

CD4+ T-cells are critical for host defense against Pneumocystis infection (43, 44, 45, 46). 463

Our previous studies have shown that during Pneumocystis infection, the utilization of peripheral 464

CD4+ T-cells increases partially due to increased destruction of these cells through enhanced 465

apoptosis (47). This acceleration of CD4+ T-cell turnover requires increased generation of CD4

+ 466

T-cells by the host. At the present time, mechanisms underlying the replenishment of CD4+ T-467


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cells remain incompletely elucidated with regard to host defense against Pneumocysitis infection. 468

No information is available regarding the significance of thymopoiesis in the host response to 469

Pneumocystis infection. 470

The results of our current investigation show that blocking thymopoiesis in adult 471

C57BL/6 mice by thymectomy delayed clearance of Pneumocystis from the lung following 472

intratracheal inoculation. These data indicate that thymic function constitutes an important 473

component of host defense against Pneumocystis infection. It has been known that HIV infection 474

and corticosteroid therapy both cause thymic toxicity. Thymic suppression in 475

immunocompromized patients particularly those with AIDS or receiving long-term 476

corticosteroid therapy may further impede host defense against Pneumocystis infection. Our mice 477

with thymectomy, however, were eventually able to eradicate Pneumocystis infection in the lung, 478

which suggests that peripheral clone expansion of existing T-cells in normal hosts can still 479

sustain host defense against Pneumocystis infection although in an inefficient manner. Our 480

current study is in agreement with clinical observations in which no increase in incidence of 481

Pneumocystis infection has been reported in individuals previously thymectomized for 482

myasthenia gravis (17). 483

In our murine model, immature thymocyte proliferation in the thymus and thymic output 484

of naïve CD4+ T-cell were significantly enhanced following Pneumocystis infection. The 485

numbers of ETPs and DN thymocytes in the thymus of sham operated mice were significantly 486

increased after intratracheal inoculation of Pneumocystis. Similarly, the level of msjTRECs in 487

CD4+ splenocytes was significantly elevated in sham operated mice at 5 weeks post 488

Pneumocystis challenge. These results support an activation of thymopoietic activity in adult 489

mice in response to Pneumocysitis infection. Interestingly, the level of msjTRECs in CD4+ 490


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splenocytes was not increased during the initial 4 weeks post Pneumocystis inoculation. A 491

possible explanation is that newly released naïve CD4+ T-cells from the thymus may be 492

preferentially recruited into the infected tissue site, i.e. the alveolar space. Our results 493

demonstrated that the numbers of naïve CD4+ T-cells and total CD4

+ T-cells recruited into lung 494

tissue were markedly increased following Pneumocystis infection. During this period of time, 495

peripheral lymphoid tissues including the spleen and lung associated lymph nodes primarily 496

produce memory T-cells through enhanced clonal expansion. Increase in homing of naïve CD4+ 497

T-cells to these peripheral lymphoid tissues may continue after clearance of the inoculated 498

Pneumocystis from the lung. Mechanisms underlying this dynamic homing of naïve CD4+ T-499

cells during the host response to Pneumocystis infection remain to be explored. Mice with 500

thymectomy showed a significant reduction of naïve CD4+ T-cells recruitment into the lung 501

following Pneumocystis infection, which suggest that pulmonary recruitment of these T-cells 502

was impaired in the absence of appropriate thymopoietic support. In week 5 of Pneumocystis 503

infection, the increase in central memory and effector memory CD4+ T-cells in the lung was also 504

attenuated in mice with thymectomy. It remains to be defined if this reduction of memory CD4+ 505

T-cells in the lung resulted directly from insufficiency of naïve CD4+ T-cell conversion or 506

involved other mechanisms yet to be identified. 507

Phenotypic analysis of CD4+ T-cells in the peripheral lymphoid tissues and spleen show a 508

decrease in the numbers of naïve and central memory CD4+ T-cells but preservation of effector 509

memory T-cells following thymectomy. These alterations of CD4+ T-cell subtypes support the 510

important role of the thymus in supporting naïve and central memory cells within lymphoid 511

tissue following a Pneumocystis challenge. These observations also provide supporting evidence 512

for the possible dynamic homing of these cells to an infected tissue site versus peripheral 513


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lymphoid tissues. 514

The active thymopoietic response to Pneumocystis infection requires thymopoietic 515

progenitors supplied by the bone marrow. Previous studies have shown that marrow 516

CCR9+MPPs represent a major precursor population of ETPs (24). Our current results show that 517

the marrow pool of CCR9+MPPs was expanded following Pneumocystis infection. This increase 518

in the number of marrow CCR9+MPPs was accompanied by the expansion of the upstream HSC 519

pool as well as the entire LKS cell population in the bone marrow. With the increase in marrow 520

CCR9+MPPs, bone marrow release of these precursors into the systemic circulation was 521

enhanced. As the consequence, the number of CCR9+MPPs in the peripheral blood was 522

significantly increased. In contrast to the increase in generation of thymopoietic precursors, the 523

numbers of lin-c-kit

+Sca-1

- cells and MPPs in the bone marrow were reduced, which suggests the 524

polarization of marrow lineage support toward T-cell production following Pneumocystis 525

infection. These data identify the bone marrow as a key component of the thymopoietic response 526

to Pneumocysitis infection. In our in vivo experiments, the levels of lin-c-kit

+Sca-1

- cells and 527

MPPs were elevated in the blood stream during the early stage of Pneumocystis infection, which 528

suggests an activated mobilization of these cells into the systemic circulation. The significance of 529

this temporarily enhanced mobilization of lin-c-Kit

+Sca-1

- cells and MPPs into the circulation 530

following Pneumocystis infection remains to be elucidated. One possible speculation for release 531

of these precursors from the bone marrow at the early stage of Pneumocystis infection is that it 532

may facilitate appropriate niche space for rapid expansion of the thymopoietic precursor cell 533

population in the bone marrow. 534

Previous studies have demonstrated that bone marrow hematopoietic precursor cells 535

express TLRs enabling these cells to respond to TLR ligand stimulation (14, 32, 52). 536


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Pneumocystis is a fungal pathogen. Its cell components including glycoprotein and (1-3)-く-D-537

glucan can be recognized by TLRs, such as TLR-2 and TLR-4 (9, 50, 52, 54). Prior 538

investigations have not addressed TLR9 in Pneumocystis, although this pattern receptor is 539

important in recognition of other fungal pathogens (39). Clinical investigations and experimental 540

studies have repeatedly shown that circulating levels of Pneumocystis derived cell wall 541

components are increased following pulmonary infection with Pneumocystis (7, 8, 53). In 542

preliminary studies, we also observed an increase in plasma concentration of (1-3)-く-D-glucan in 543

mice with Pneumocystis pneumonia (data not shown). Our current investigation shows that after 544

in vitro exposure to Pneumocystis extracts, the number of LKS cells, HSCs, MPPs, CCR9+MPPs, 545

and CLPs were significantly increased in cultured bone marrow cells. Culture of bone marrow 546

cells with TLR-2 ligand Zymosan, TLR-4 ligand LPS, and TLR-9 ligand ODN M362 resulted in 547

a similar response. These data suggest that TLR signaling may be involved in mediating the 548

alteration of marrow hematopoietic precursor cell repertoire during Pneumocystis infection. 549

Recent studies from our group have revealed that marrow lin-c-Kit

+Sca-1

- cells can 550

convert to LKS cells through re-expression of Sca-1 in response to infectious stimuli (56). This 551

phenotypic conversion of the downstream progenitors to upstream LKS cells serves as a major 552

mechanism underlying the rapid expansion of marrow LKS cell pool. In our current investigation, 553

we also observed that in association with the increase in marrow LKS cell and CCR9+MPP 554

populations, the number of lin-c-Kit

+Sca-1

- cells in the bone marrow was reduced in mice 555

following Pneumocystis infection. Furthermore, culture of bone marrow cells with Pneumocystis 556

extracts resulted in an increase in the number of LKS cells and CCR9+MPPs. Concomitantly, the 557

number of lin-c-Kit

+Sca-1

- cells was reduced in the culture system. These data support the 558

concept that up-regulation of Sca-1 in marrow precursor cells plays a critical role in 559


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reprogramming of these precursor cell lineage commitment during the host response to 560

Pneumocystis infection. In studies previously reported by our group, we have observed that mice 561

with E. coli bacteremia also show a rapid expansion of marrow LKS cell population (56). 562

Phenotypic conversion of lin-c-kit+Sca-1- cells to LKS cells by expression of Sca-1 plays a 563

predominant role in the expansion of marrow LKS cell pool. This marrow LKS cell response 564

forms a platform for reprogramming hematopoietic precursor cell lineage commitment toward 565

granulocyte production in the process of host defense against bacterial infection. These 566

observations suggest that the host defense response to infectious pathogens may share a similar 567

mechanism at the primitive hematopoietic precursor level. However, reprogramming of these 568

precursor cells for selected lineage commitment relies on unique signaling generated from 569

infection with a specific pathogen type. 570

By mapping the promoter region of the Sca-1 gene, we observed that the Sca-1 promoter 571

region contains multiple AP1 binding sites. C-Jun is the most potent transcriptional activator in 572

the AP1 family (28). Studies have previously shown that engagement of TLR-2, TLR-4, and 573

TLR-9 with their ligands all activate JNK leading to enhancement of c-Jun transcriptional 574

activity by phosphorylation of its N-terminal activation domain (14). Therefore, we determined if 575

the JNK signal pathway was involved in mediating the hematopoietic precursor cell response to 576

Pneumocystis infection. The results of our experiments showed that Pneumocystis extracts, 577

Zymosan, and ODN M362 each activated JNK in cultured bone marrow cells. Addition of 578

specific JNK inhibitor SP600125 to the culture system profoundly inhibited the increase in LKS 579

cells and CCR9+MPPs in cultured bone marrow cells following exposure to Pneumocystis 580

extracts, Zymosan, or ODN M362. These findings demonstrate that the TLR-JNK-AP-1 581

signaling cascade may play a vital role in mediating the enhancement of marrow T-cell lineage 582


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support during the host defense response to Pneumocystis infection. 583

In our in vivo experiments, the plasma levels of IL-3, IL-7, IL-9, IFN-け, and FLT3 lignad 584

were not altered. Previous investigations have shown that these mediators may modulate 585

thymopoietic activity. However, the role of these mediators (if any) in our model of 586

Pneumocystis infection remains undetermined. 587

In summary, thymopoietic activity is enhanced following Pneumocystis pneumonia in 588

adult C57Bl/6 mice along with increases in thymopoietic precursor cells in the bone marrow. 589

Ready access of Pneumocystis-derived TLR ligands into the systemic circulation may stimulate 590

bone marrow primitive hematopoietic precursor cell reprogramming to enhance their T 591

lymphocyte lineage commitment through activation of the JNK-AP-1 pathway. 592


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Acknowledgments 593

We thank Dr. Joseph S. Sblosky, Ms. Connie P. Porretta, Ms. Jane A. Schexnayder, and 594

Ms. Amy B. Weinberg for their technical assistance. 595

This work was supported by National Institutes of Health Grants: HL076100, AA017494, 596

AA019676, and AG25150. 597

598


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Cells. 26:1778-86. 764

765

766

767

768

769

770

771


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772

773

774

775

TABLE 1. Markers of cell types (1-3, 13, 18-21, 23-26, and 34). 776

______________________________________________________________________________ 777 LKS cells Lin

-c-kit

+Sca-1

+ 778

HSCs: Lin-c-kit

+Sca-1

+ Thy-1.1

loCD34

- 779

MPPs: Lin-c-kit

+Sca-1

+Thy-1.1

-CD34

+ 780

CCR9+MPPs: Lin

-c-kit

+Sca-1

+Thy1.1

- CD34

+CCR9

+VCAM1

- 781

CLPs: Lin-c-kit

loSca-1

lo Thy1.1

- IL-9Tg"+ 782

Lin-c-kit

+Sca-1

- cells Lin

-c-kit

+Sca-1

- 783

CMPs: Lin-c-kit

+Sca-1

-IL-9Tg-

784

ETPs: Lin(CD19, CD3,CD4,CD8, F4-80, Gr-1)- 785

c-kit+Sca-1

+CD44

+CD25

- 786

DN1 cells: CD4-CD8

-CD44

+CD25

- 787

DN2 cells: CD4-CD8

-CD44

+CD25

+ 788

DN3 cells: CD4-CD8

-CD44

-CD25

+ 789

DN4 cells: CD4-CD8

-CD44

-CD25

- 790

791

CD4+ T-cells: CD3

+CD4

+ 792

Naïve CD4+ T-cells: CD3

+CD4

+CD44

loCD62L

hi 793

Central memory CD4+

T-cells: CD3+CD4

+CD44

hiCD62L

hi 794

Effect memory CD4+

T-cells: CD3+CD4

+CD44

hiCD62L

lo 795

_____________________________________________________________________________________________________________________ 796

797

798

799

800

801

802

803


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804

805

806

807

808

809

TABLE 2. Circulating lymphocytes / 106

WBCs 810

Total CD4+ cells Naïve CD4

+ cells 811

Sham Operation Thymectomy Sham Operation Thymectomy 812

Control 166840 ± 12486 84688 ± 5509Ä 33180 ± 2105 10699 ± 784

Ä 813

Week 1 167887 ± 15814 122379 ± 5967Ä

33609 ± 3005 13987 ± 1447Ä

814

Week 2 173765 ± 7100 86929 ± 5260Ä 33804 ± 2656 8420 ± 1390

Ä 815

Week 3 149101 ± 6150 80327 ± 5050

Ä 27040 ± 2265 6217 ± 803

Ä 816

Week 4 167127 ± 10107 90098 ± 4421Ä 32886 ± 2167 6026 ± 334

Ä 817

Week 5 163342 ± 5947 69941 ± 2772Ä 30048 ± 1761 4580 ± 480

Ä 818

Week 6 189010 ± 14427 89827 ± 6937Ä 28823 ± 909 5222 ± 707

Ä 819

820

Central memory CD4+ cells Effector memory CD4

+ cells 821

Sham Operation Thymectomy Sham Operation Thymectomy 822

Control 99708 ± 6326 52921 ± 5037Ä 27043 ± 4260 18618 ± 3226 823

Week 1 105213 ± 10083 72511 ± 3672Ä

26796 ± 3220 34073 ± 1969 824

Week 2 100409 ± 4443 47152 ± 3482Ä 36292 ± 1639 30035 ± 1640

825

Week 3 90799 ± 4113 46808 ± 3350Ä 29303 ± 1911 26314 ± 1400

826

Week 4 97448 ± 6530 48584 ± 2836Ä 34237 ± 2555 34555 ± 2111

827

Week 5 93265 ± 4370 37048 ± 2632Ä 37685 ± 2715 27432 ± 3352

828

Week 6 101725 ± 9718 37742 ± 6323Ä 54749 ± 4394* 45439 ± 3019

829

Mice with or without thymectomy were challenged with intratracheal Pneumocystis (2 x 105 830

cysts/mouse). A blood sample was collected each week after the Pneumocystis infection. CD4+ 831

cell types were determined by flowcytometry. N=5. *and : p < 0.05 vs. the corresponding 832

control group (uninfgevgf"okeg+="Ä<"p < 0.05 vs. sham operated mice at same time point. 833

834

835


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Figure Legends 836 837

Figure 1. Copies of Pneumocystis rRNA in the whole lung. N=5. *: p < 0.05 vs. week 1 838

sham operated mice. ﾒ: p < 0.05 vs. sham operated mice at same time point. 839

Figure 2. CD4+ cell types recovered from bronchoalveolar lavage fluid (BALF). N=5. * 840

and à: p < 0.05 vs. the corresponding control group (uninfected mice). 剏: p < 841

0.05 vs. sham operated mice at same time point. 842

Figure 3. CD4+ cell types isolated from lung-associated lymph nodes (3A) and spleen (3B). 843

N=5. * and ┇: p < 0.05 vs. the corresponding control group (uninfected mice). : 844

p < 0.05 vs. sham operated mice at same time point. 845

Figure 4. The number of earliest thymic progenitors (ETPs) (4A) and double negative (DN) 846

cells (4B) in the thymus. N=5. *, à, , and ^: p< 0.05 vs. the corresponding cell 847

type in the control group (uninfected mice). 848

The level of murine single joint T cell receptor excision circles (msjTRECs) DNA 849

in CD4+ splenocytes (4C). N=5. *: p < 0.05 vs. the corresponding control group 850

(uninfected mice). : p < 0.05 vs. sham operated mice at same time point. 851

Figure 5. Hematopoietic precursor cell types in the bone marrow (5A) and circulation (5B). 852

N=5. * and à: p<0.05 vs. the corresponding control group (uninfected mice). : 853

p<0.05 vs. sham operated mice at same time point. 854

Figure 6. Changes in bone marrow cell types following culture with different stimuli. N=5. 855

*: p<0.05 vs. the corresponding control group. 856


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Figure 7. Changes in the number of CCR9+MPPs and LKS cells in bone marrow cells 857

following culture with different doses of TLR-2 and TLR-9 ligands. N=5. *: 858

p<0.05 vs. the corresponding control group. 859

Figure 8. Phospho-JNK level in nucleated bone marrow cells following culture with TLR 860

ligands (8A). The image is a representative of 4 sets of cultures. *: p<0.05 vs. 861

control group. 862

The effects of JNK inhibitor SP600125 (20 µM) on alteration of bone marrow cell 863

types following culture with different stimuli (8B). N=5. *: p<0.05 vs. control 864

group; ┇: p<0.05 vs. cells cultured without SP600125. 865


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