1 Increased ferroportin-1 expression and rapid splenic iron loss occur with anemia caused by 1 ...

37 Pages·2015·1.3 MB·English

IAI Accepted Manuscript Posted Online 30 March 2015
Infect. Immun. doi:10.1128/IAI.02863-14
Copyright © 2015, American Society for Microbiology. All Rights Reserved.

1 Increased ferroportin-1 expression and rapid splenic iron loss occur with anemia caused by
2 Salmonella infection in mice
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4 Running Title - Ferroportin-1 and Salmonella Infection
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6 Diane E. Brown1,2#, Heidi J. Nick3, Melissa W. McCoy3,4, Sarah M. Moreland3,5, Aaron M.
7 Stepanek2, Ross Benik6, Karyn E. O’Connell7, Maria C. Pilonieta3, Toni A. Nagy3, Corrella S.
8 Detweiler3#
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10 1Department of Pathology, Massachusetts General Hospital and Harvard Medical School,
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11 Boston, MA USA o
12 2Center for Comparative Medicine, Massachusetts General Hospital, Boston, MA USA w
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13 3Department of Molecular, Cellular and Developmental Biology, University of Colorado, lo
14 Boulder, CO USA a
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15 4Present address: Association of Clean Water Administrators, 1221 Connecticut Ave NW, e
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16 Washington, D.C. USA
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17 5Present address: School of Medicine, Temple University, Philadelphia, PA, USA ro
18 6Premier Laboratories, Boulder, CO USA m

19 7Cancer Vaccine Center, Department of Medical Oncology, Cancer Vaccine Center, Dana Farber h
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20 Cancer Institute, Boston, MA USA p
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23 Statement of Equal Authors’ Contributions: a
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24 DEB and CSD contributed equally to this manuscript. m
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26 #Corresponding authors: rg
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27 D. Brown, [email protected]; C. Detweiler, [email protected] o
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28
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29 Key words: Salmonella, macrophage, FPN1, anemia, iron, inflammation p
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31 Abbreviations: S. Typhimurium, CFU, EMH, Fpn1, IFNγ, MCP-1, TNFα 1
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33 Abstract
34 The Gram-negative intracellular bacterium Salmonella enterica serovar Typhimurium causes
35 persistent systemic inflammatory disease in immunocompetent mice. Following oral inoculation
36 with S. Typhimurium, mice develop a hematopathological syndrome akin to typhoid fever with
37 splenomegaly, microcytic anemia, extramedullary erythropoiesis, and increased hemophagocytic
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38 macrophages in the bone marrow, liver and spleen. Additionally, there is marked loss of iron
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39 from the spleen, an unanticipated result given the iron sequestration reported in anemia of lo
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40 inflammatory disease. To establish why tissue iron does not accumulate, we evaluated multiple d

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41 measures of pathology for four weeks following oral infection in mice. We demonstrate a m

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42 quantitative decrease in splenic iron following infection despite increased numbers of splenic t
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43 phagocytes. Infected mice have increased duodenal expression of the iron exporter ferroportin-1, ia
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44 consistent with increased uptake of dietary iron. Liver and splenic macrophages also express s
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45 high levels of ferroportin-1. These observations indicate that splenic and hepatic macrophages r
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46 export iron during S. Typhimurium infection, in contrast to macrophage iron sequestration n

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47 observed in anemia of inflammatory disease. Tissue macrophage export of iron occurs p
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48 concurrent with high serum concentrations of IFNγ and IL-12. In individual mice, high serum 2
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49 concentrations of both pro-inflammatory (TNFα) and anti-inflammatory (IL-10) cytokines 0
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50 correlate with increased tissue bacterial loads throughout four weeks of infection. These in vivo b
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51 observations are consistent with previous cell culture studies and suggest that the re-location of u
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52 iron from tissue macrophages during infection may contribute to anemia and also to host survival
53 of acute S. Typhimurium infection.
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54 Introduction
55 Typhoid fever is acquired upon oral ingestion of food or water contaminated with
56 Salmonella enterica serovars Typhi or Paratyphi and remains a serious threat to public health,
57 especially in developing countries (1). Human typhoid fever causes a broad range of clinical
58 signs including splenomegaly, neuropathy and hematopathology such as cytopenias,(2) that have
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59 been modeled in laboratory mice infected with Salmonella enterica serovar Typhimurium (S.
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60 Typhimurium) (3). Inbred mouse strains of differing genetic backgrounds are characterized as lo
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61 either sensitive or resistant to S. Typhimurium. Sensitivity to this intracellular bacterial infection d

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62 is caused by genetic deficiencies in the innate or adaptive immune system (4, 5). For instance, m

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63 mice lacking the cation transporter Nramp1 (Slc11a1), a regulator of cellular iron metabolism, t
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64 are extremely sensitive to infection. Nramp1+ mice are considered resistant to S. Typhimurium ia
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65 because infection is generally non-fatal (6–8). Resistant mice infected by natural oral inoculation s
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66 develop systemic infection with an acute hematopathological syndrome similar to that of humans r
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67 with typhoid fever, including fever, anemia, inflammatory disease characterized by peripheral n

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68 blood neutrophilia and monocytosis, bone marrow myeloid hyperplasia and hemophagocytosis, p
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69 and splenomegaly secondary to extramedullary hematopoiesis (EMH) and phagocyte infiltration 2
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70 (7, 8). The bacteria reside within tissue macrophages (6, 9) and hemophagocytic macrophages 0
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71 (10), which have phagocytosed intact erythrocytes and leukocytes. Recently, it has been b
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72 observed that S. Typhimurium acquires iron from hemophagocytes (11). u
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73 During inflammation, macrophages typically sequester iron, a process that reduces iron
74 availability in serum (12). Macrophage sequestration of iron is a form of ‘nutritional immunity’
75 because most bacteria require iron to replicate (13–15). However, infections with intracellular
76 pathogens such as S. Typhimurium and Mycobacterium tuberculosis appear to represent a special
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77 case. For instance, cell culture macrophages infected with S. Typhimurium restrict pathogen iron
78 access by increasing expression of the iron transporter ferroportin-1 (Fpn1) (16, 17). Fpn1 is the
79 only known eukaryotic cellular iron exporter (18) and as such is a major contributor to host iron
80 regulation. Fpn1 expression is induced by nitric oxide, which in turn is induced by the pro-
81 inflammatory cytokines interferon-gamma (IFNγ) (19) and tumor necrosis factor alpha (TNFα)
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82 (20). In cell culture, IFNγ treatment of S. Typhimurium-infected macrophage-like cells increases
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83 Fpn1 expression while reducing cellular iron content and inhibiting bacterial replication (16). lo
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84 Further experiments show Fpn1-mediated control of intracellular S. Typhimurium growth in d

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85 cultured murine macrophages and HeLa cells (21). However, the relationship of these cellular m

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86 observations to in vivo pathology is not known. t
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87 We previously demonstrated that during the acute phase response to inflammation, ia
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88 resistant (Nramp1+) mice infected with S. Typhimurium by the natural oral route develop s
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89 decreased serum iron, increased serum ferritin, and increased hemophagocytosis in bone marrow, r
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90 liver and spleen. Infected mice develop microcytic anemia that is most severe one to three weeks n

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91 post-infection and persistent for nine weeks along with erythrocyte fragmentation and severely p
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92 diminished tissue iron staining from one to six weeks post-infection (7, 8). These 2
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93 hematopathological findings are characteristic features of systemic iron deficiency (22). The 0
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94 observation of decreased tissue iron in infected, anemic mice in the face of marked inflammatory b
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95 disease was unexpected based on the well-described macrophage iron sequestration that occurs u
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96 with anemia of inflammatory (or chronic) disease (AID) (23). Our aim was to quantitate and
97 establish mechanisms by which tissue iron declines in mice infected with the intracellular
98 pathogen, S. Typhimurium, by monitoring measures of iron metabolism, cellular infiltrates and
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99 cytokine responses over an infection course that is acute to subacute (between acute and chronic
100 in character (24)).
101 Methods
102 Bacterial Cultures - Freshly struck colonies of virulent S. Typhimurium strain SL1344 (StrR)
103 (25), inoculated into Luria-Bertani broth with streptomycin (200mg/mL) were grown at 37°C
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104 with aeration overnight. Bacteria were pelleted and re-suspended in sterile PBS.
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105 lo
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106 Animals - Research protocols were in accordance with the NIH Guide for the Care and Use of d

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107 Laboratory Animals, and approved by the University of Colorado Institutional Biosafety and m

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108 Animal Care and Use Committees. Seven to eight-week-old Sv129S6/SvEvTac (Sv129) female t
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109 and male mice (Taconic Farms, Inc.; Hudson, NY) were bred in-house. Mice received 2918 ia
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110 Teklad Global 18% Protein Rodent Diet (Harlan Laboratories). This chow contains iron at 200 s
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111 ppm, well above the minimum daily requirement (35 ppm; (26)) but below what is considered a r
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112 high iron diet of 300 ppm (27). Mice were fasted for 2-4 hours prior to infection via oral gavage n

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113 with 1x109 colony forming units (CFU) S. Typhimurium in 100 µL PBS, as determined by p
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114 plating. Control mice were mock-infected with 100 µL PBS, and housed separately. Mice were 2
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115 euthanized and evaluated at 1, 2, 3, and 4 weeks post-infection (repeated experiments). Numbers 0
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116 of animals are indicated in figure legends. b
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118 Blood and Tissue Collection and Processing - Blood was collected by cardiocentesis
119 immediately following carbon dioxide asphyxiation. Following euthanasia whole spleen and
120 liver were weighed; a section of each was weighed, homogenized and plated for bacterial CFU
121 per gram of tissue. No CFU were found in any mock-infected animals. Remaining liver and
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122 spleen, and one femur were collected into 10% neutral buffered formalin. Bone marrow brush
123 preparations, made immediately post-mortem from the second femur were Wright-stained for
124 cytological evaluation. Tissues were processed by routine histological methods; paraffin sections
125 were stained with hematoxylin and eosin (H & E) and Perl’s Prussian blue for ferric iron
126 (hemosiderin). Intensity of iron tissue staining was subjectively scored in a blinded fashion by a
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127 board-certified veterinary pathologist (DEB) using a scale of 0 – 4+ by light microscopy. All
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128 photomicrographs were taken with an Olympus BX50 (Olympus Corporation, Center Valley, lo
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129 PA) using QImagingMicroPublisher RTV 5.0 (JH Technologies, Fremont, CA). d

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130 m

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131 Immunohistochemistry - All tissue sections for immunohistochemistry were formalin-fixed, t
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132 paraffin-embedded, sectioned at 3 microns, baked at 60°C for 1 hour, de-paraffinized and re- ia
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133 hydrated, and pretreated with proteinase K for antigenic retrieval, then treated specifically as s
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134 described below for each antibody. All sections were reviewed in a blinded fashion by a board- r
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135 certified veterinary pathologist (DEB). n

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136 p
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137 Ferroportin 1 (Fpn1)/Prussian blue: To quench endogenous peroxidase activity, sections were 2
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138 treated with 3.0% hydrogen peroxide solution for 5 minutes, at room temperature (RT). 0
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139 Enzymatic pretreatment with proteinase K (Dako S3020) for 5 minutes (RT) was used for b
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140 antigenic retrieval. Serum Free Protein Block (Dako, X0909) was used at 5 minutes (RT) to u
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141 neutralize any charged molecules on the tissue sections that may cause non-specific staining.
142 Rabbit anti-mouse Fpn1 antibody (Lifespan Biosciences, LS-C16620) was prepared at a working
143 dilution of 0.02 mg/mL by diluting the stock solution in antibody diluent (Dako S0809). Fpn1
144 antibody was incubated on tissue sections for 30 minutes (RT). A negative control was incubated
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145 with rabbit IgG isotype solution (Calbiochem NI01) at a concentration of 0.02 mg/mL at the
146 same duration and temperature as the primary antibody. The Fpn1 antibody was then conjugated
147 with an Envision+HRP rabbit polymer (Dako K4003) for 30 minutes, RT. Staining was
148 developed with a DAB+ chromogen system (Dako K3468) for 5 minutes, RT. The detection of
149 ferric iron was accomplished by following a routine Prussian blue histological staining method.
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150 The Prussian blue working solution was prepared by mixing equal parts of 2.0% hydrochloric
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151 acid and 2.0% potassium ferrocyanide then preheated to 60°C. The slides were incubated in the lo
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152 Prussian blue working solution for 30 minutes at 60°C. Slides were finally counter-stained in d

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153 nuclear fast red for 5 minutes, RT. m

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154 t
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155 F4/80: To quench endogenous alkaline phosphatase activity, a Duel Endogenous Enzyme Block ia
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156 solution (Dako, S2003) was treated on the tissue sections for 10 minutes (RT). Enzymatic s
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157 pretreatment with proteinase K (Dako, S3020) for 5 minutes (RT) was used for antigenic r
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158 retrieval. Serum Free Protein Block (Dako, X0909) was used at 5 minutes (RT) to neutralize any n

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159 charged molecules on the tissue sections that may cause non-specific staining. Rat anti-mouse p
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160 F4/80 antibody (Serotec, MCA497) was prepared at a working dilution of 1:300 by diluting the 2
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161 stock solution in antibody diluent (Dako, S0809). F4/80 antibody was incubated on tissue 0
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162 sections for 30 minutes (RT). The negative control section was incubated with rat IgG2b isotype b
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163 solution (Serotec, MCA1125) at the same duration, concentration and temperature as the primary u
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164 antibody (approximately 0.166 µg/mL). F4/80 antibody was then conjugated with biotinylated
165 rabbit anti-rat Immunoglobulin (Dako E0468) for 30 minutes (RT). The secondary antibody was
166 then enhanced with an anti-rabbit-alkaline phosphatase labeled polymer (BioCare Medical,
167 RALP525L) for 30 minutes (RT). Staining was developed with a red chromogen system
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168 (BioCare Medical, FR805S) for 10 minutes (RT). Counter staining was performed with
169 hematoxylin for 10 minutes (RT). The positive control tissue used was a mouse tissue xenograph
170 injected with a human HT-29 tumor cell line (data not shown).
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172 iNOS: Anti-iNOS (1:100, Abcam, Cambridge, MA) and controls were run in parallel and
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173 included secondary-only and isotype serum (rabbit IgG, Santa Cruz Biotechnology, Santa Cruz,
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174 CA). Slides were blocked in donkey serum in PBS-Triton X for 45 minutes, incubated with lo
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175 primary antibodies diluted in PBS overnight at 4°C, washed with PBS and incubated with d

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176 donkey anti-rabbit AlexaFluor® 594 (Invitrogen, Grand Island, NY) for 30 minutes at 37°C in m

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177 the dark, then coverslipped with Vectashield Mounting Media with DAPI (Vector Laboratories, t
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178 Inc. Burlingame, CA). All sections were photographed at 400x (40x objective) using identical ia
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179 exposure times. m
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180 rg
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181 Real-time reverse transcription PCR n

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182 Spleen and liver were harvested from mock- or S. Typhimurium-infected mice at 1 and 3 weeks r
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183 post-infection. A portion of each was weighed, placed into >10 µl of RNAlater (Qiagen) per 1 2
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184 mg of tissue, and stored at -80°C until subsequent isolation of total RNA using the RNeasy Midi 1
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185 Kit (Qiagen), including on-column DNase treatment, according to the manufacturer’s y

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186 instructions. First-strand cDNA was synthesized from 250 ng of total RNA using the iScript e
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187 cDNA Synthesis Kit (Bio-Rad, 170-8891; blend of oligo(dt) and random hexamers) according to
188 the manufacturer’s protocol. The cDNA was subsequently diluted 4-fold (iNOS) or 20-fold
189 (hepcidin and Fpn1) with nuclease-free water. TaqMan qRT-PCR assays were carried out using
190 the following pre-designed primer/probe sets: Hprt (VIC-MGB probe, primer limited, Invitrogen
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191 4448484, assay ID Mm00446968_m1); hepcidin (Hamp1, Invitrogen 4331182, assay ID
192 Mm04231240_s1, detects Hamp1 only); Fpn1 (FAM-MGB probe, Invitrogen 4331182, assay ID
193 Mm01254822_m1); or iNos (Nos2, FAM-MGB probe, Invitrogen 4331182, assay ID
194 Mm00440502_m1). Selection of Hprt for normalization was based on validation experiments
195 which determined that splenic and hepatic Hprt expression was not significantly different for
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196 mock versus infected mice. qRT-PCR reaction mixtures (20 µl) contained 8 µl of 4- or 20-fold
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197 diluted cDNA, 10 µl of TaqMan Universal PCR Master Mix II with UNG (Invitrogen, 4440038), lo
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198 1 µl of Hprt primer/probe and 1 µl target gene primer/probe or water. Hamp1 and Fpn1 d

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199 expression assays were multiplexed with Hprt, while iNos expression assays were singleplexed m

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200 due to interference between the iNos and Hprt primer/probe sets as determined by validation and t
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201 optimization experiments. Reactions were run on a CFX384 real-time PCR detection system ia
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202 (Bio-Rad) under the following cycling conditions: 2 min at 50°C, 10 min at 95°C, then 45 cycles s
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203 at 95°C for 15 sec and 60°C for 60 sec. No-RNA and no-reverse transcriptase controls were r
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204 included for each gene assayed. Amplification results were baseline- and drift-corrected using n

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205 the CFX Manager software, followed by manual adjustment of the Cq threshold for each p
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206 fluorophore. Data were then exported to Microsoft Excel for further analysis. Relative gene 2
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207 expression was determined according to the Livak method (28) as follows: 1) Fpn1, iNos, and 0
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208 Hamp1 expression for each sample were normalized to Hprt (ΔCt); 2) On a tissue- and time b
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209 point-specific basis, the mean of the target gene ΔCts of the control mice group was set as the u
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210 calibrator for calculation of the ΔΔCt values for each sample; 3) The fold difference in target
211 gene expression for each sample was then calculated using the 2-ΔΔCt equation. Reaction
212 efficiencies for each primer/probe set were assessed by performing real-time PCR on serial 10-
213 fold dilutions of cDNA, plotting the Ct values for each dilution against the log of the input
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214 concentration, and determining the efficiency using the equation, efficiency = -1 + 10(-1/slope).
215 Statistical analysis of the mean fold changes between groups of mock and infected mice was
216 carried out using one-way ANOVA followed by Tukey’s multiple comparisons test (GraphPad
217 Prism version 5.00).
218
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219 Flow Cytometry - Portions of spleens were mechanically homogenized and passed through a 70
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220 µM cell strainer. Non-nucleated erythrocytes in aliquots of each suspension were lysed in lo
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221 hypotonic buffer containing 0.16 M NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA, and remaining d

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222 cells were then counted on a hemocytometer to estimate the number of nucleated cells per m

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223 spleen. Additional aliquots were lysed in PBS with 0.2% NP-40 and plated for bacterial CFU. t
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224 Unlysed cells were washed in RPMI, passed through a 40 µM cell strainer and re-suspended in ia
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225 staining buffer (PBS + 1% fetal bovine serum and 0.02% sodium azide) containing anti-mouse s
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226 CD16/32 (eBioscience; San Diego, CA) to block Fc receptors. Cells were then stained with r
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227 phycoerythrin-conjugated anti-mouse CD68 (AbDSerotec; Raleigh, NC), allophycocyanin- n

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228 conjugated anti-mouse CD11c (eBioscience; San Diego, CA) and phycoerythrin-Cy7-conjugated p
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229 anti-mouse Gr-1 (Ly-6G, eBioscience; San Diego, CA), fixed in 1% paraformaldehyde/1% 2
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230 sucrose, permeabilized in 0.1% saponin, and stained for DNA in 10 µg/ml DAPI in buffer 0
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231 containing 0.1% saponin. Fluorescently labeled cells were quantified using a CyAn ADP flow b
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232 cytometer (Beckman Coulter; Indianapolis, IN) and analyzed using FlowJo software (Tree Star; u
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233 Ashland OR). Macrophages were identified as CD68+, CD11clow/- (αX integrin, a dendritic cell
234 marker) and Gr-1int/- (a monocyte and granulocyte/neutrophil marker). Dendritic cells were
235 identified as CD11chigh, neutrophils as Gr-1high, and monocytes as Gr-1int, CD11clow/-, and CD68-
236 (29). The number of granulocytes, monocytes, macrophages and dendritic cells per spleen was
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