The role of intracellular thyroid hormone metabolism in innate immune cells

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van der Spek, A.H.

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2018

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van der Spek, A. H. (2018). The role of intracellular thyroid hormone metabolism in innate

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TYPE 3 DEIODINASE IS ESSENTIAL FOR

OPTIMAL NEUTROPHIL FUNCTION -

OBSERVATIONS FROM 3 SPECIES

Anne H. van der Spek, Kin Ki Jim, Aldona Karaczyn, Hermina C. van Beeren,

Mariëtte T. Ackermans, Veerle M. Darras, Christina M.J.E. Vandenbroucke-Grauls,

Arturo Hernandez, Matthijs C. Brouwer, Eric Fliers, Diederik van de Beek†, Anita Boelen†

*/† these authors contributed equally to this work

Endocrinology 2018 Feb 1;159(2):826-835.

TYPE 3 DEIODINASE IS ESSENTIAL FOR

OPTIMAL NEUTROPHIL FUNCTION -

OBSERVATIONS FROM 3 SPECIES

Anne H. van der Spek, Kin Ki Jim, Aldona Karaczyn, Hermina C. van Beeren,

Mariëtte T. Ackermans, Veerle M. Darras, Christina M.J.E. Vandenbroucke-Grauls,

Arturo Hernandez, Matthijs C. Brouwer, Eric Fliers, Diederik van de Beek†, Anita Boelen†

*/† these authors contributed equally to this work

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72

Abstract

Neutrophils are essential effector cells of the innate immune system that have recently been recognized as thyroid hormone (TH) target cells. Cellular TH bioavailability is regulated by the deiodinase enzymes, which can activate or inactivate TH. We have previously shown that the TH inactivating enzyme type 3 deiodinase (D3) is present in neutrophils. Furthermore, D3KO mice show impaired bacterial killing upon infection. We hypothesized that D3 plays a role in neutrophil function during infection by actively regulating local TH availability.

We measured TH concentrations in cerebrospinal fluid (CSF) from patients with bacterial meningitis and controls. Bacterial meningitis resulted in marked changes in CSF TH levels, characterized by a strong increase of thyroxine and reverse-triiodothyronine concentrations. This altered TH profile was consistent with elevated D3 activity in infiltrating neutrophils at the site of infection. D3 knockdown in zebrafish embryos with pneumococcal meningitis resulted in increased mortality and reduced neutrophil infiltration during infection. Finally, stimulated neutrophils from female D3KO mice exhibited impaired NADPH-oxidase activity, an important component of the neutrophil bacterial killing machinery. These consistent findings across experimental models strongly support a critical role for reduced intracellular TH concentrations in neutrophil function during infection, for which the TH inactivating enzyme D3 appears essential.

73

5 Introduction

Neutrophils are crucial for the host defense against invading pathogens. These highly specialized innate immune cells are capable of phagocytosing and killing bacteria and other microorganisms (Kolaczkowska and Kubes, 2013). Neutrophil function is of fundamental importance for survival, as illustrated by the detrimental clinical consequences of an impaired neutrophil bacterial killing machinery (Dinauer, 2014). Innate immune cells, including neutrophils, have recently been recognized as novel thyroid hormone (TH) target cells (van der Spek et al., 2017b).

TH is essential for growth and development (Brent, 2012). The thyroid gland secretes TH into the circulation, mainly in the form of the prohormone thyroxine (T4). T4 requires conversion to the active hormone triiodothyronine (T3) to exert its biological activity (Brent, 2012). The conversion of TH at the cellular and tissue level is largely regulated by the deiodinase enzymes (Gereben et al., 2008). This family of enzymes can either activate or inactive TH within the cell, thus controlling intracellular TH bioavailability. The TH inactivating enzyme type 3 deiodinase (D3) converts T4 and T3 to their inactive metabolites (reverse T3 (rT3) and 3,3’-diiodothyronine (T2), respectively) (Gereben et al., 2008). Due to its high expression in infiltrating murine and human neutrophils D3 is thought to play an important role in their function (Boelen et al., 2008, van der Spek et al., 2016, van der Spek et al., 2017b). In addition, mice that lack D3 demonstrate impaired bacterial clearance following infection (Boelen et al., 2009). These data suggest that D3 is important for bacterial killing, a hallmark of neutrophil function. We hypothesized that D3 plays a role in neutrophil function during bacterial infection by actively regulating local TH bioavailability. Here we show that bacterial meningitis results in an altered TH profile at the site of infection, consistent with elevated D3 activity in infiltrating neutrophils. In addition, a lack of D3 results in impaired survival during bacterial meningitis in a zebrafish model. Finally, neutrophils derived from D3KO mice display functional abnormalities compared to wildtype cells. Our data suggest that neutrophil function during infection requires strict control of TH availability for which D3 appears essential.

Materials and Methods

Study Approval

All animal and human experiments were conducted in compliance with institutional and (inter)national guidelines and regulations. In the case of the Dutch Bacterial Meningitis Cohort, written informed consent was obtained from all participating patients or their legally authorized representatives prior to study inclusion (Bijlsma et al., 2016).

Human subjects

CSF samples were derived from the Dutch Bacterial Meningitis Cohort which includes adults with community-acquired bacterial meningitis with positive CSF cultures. Samples were obtained from the initial diagnostic lumbar puncture. These patients were identified by the Netherlands Reference

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72

Abstract

Neutrophils are essential effector cells of the innate immune system that have recently been recognized as thyroid hormone (TH) target cells. Cellular TH bioavailability is regulated by the deiodinase enzymes, which can activate or inactivate TH. We have previously shown that the TH inactivating enzyme type 3 deiodinase (D3) is present in neutrophils. Furthermore, D3KO mice show impaired bacterial killing upon infection. We hypothesized that D3 plays a role in neutrophil function during infection by actively regulating local TH availability.

We measured TH concentrations in cerebrospinal fluid (CSF) from patients with bacterial meningitis and controls. Bacterial meningitis resulted in marked changes in CSF TH levels, characterized by a strong increase of thyroxine and reverse-triiodothyronine concentrations. This altered TH profile was consistent with elevated D3 activity in infiltrating neutrophils at the site of infection. D3 knockdown in zebrafish embryos with pneumococcal meningitis resulted in increased mortality and reduced neutrophil infiltration during infection. Finally, stimulated neutrophils from female D3KO mice exhibited impaired NADPH-oxidase activity, an important component of the neutrophil bacterial killing machinery. These consistent findings across experimental models strongly support a critical role for reduced intracellular TH concentrations in neutrophil function during infection, for which the TH inactivating enzyme D3 appears essential.

73

5 Introduction

Neutrophils are crucial for the host defense against invading pathogens. These highly specialized innate immune cells are capable of phagocytosing and killing bacteria and other microorganisms (Kolaczkowska and Kubes, 2013). Neutrophil function is of fundamental importance for survival, as illustrated by the detrimental clinical consequences of an impaired neutrophil bacterial killing machinery (Dinauer, 2014). Innate immune cells, including neutrophils, have recently been recognized as novel thyroid hormone (TH) target cells (van der Spek et al., 2017b).

TH is essential for growth and development (Brent, 2012). The thyroid gland secretes TH into the circulation, mainly in the form of the prohormone thyroxine (T4). T4 requires conversion to the active hormone triiodothyronine (T3) to exert its biological activity (Brent, 2012). The conversion of TH at the cellular and tissue level is largely regulated by the deiodinase enzymes (Gereben et al., 2008). This family of enzymes can either activate or inactive TH within the cell, thus controlling intracellular TH bioavailability. The TH inactivating enzyme type 3 deiodinase (D3) converts T4 and T3 to their inactive metabolites (reverse T3 (rT3) and 3,3’-diiodothyronine (T2), respectively) (Gereben et al., 2008). Due to its high expression in infiltrating murine and human neutrophils D3 is thought to play an important role in their function (Boelen et al., 2008, van der Spek et al., 2016, van der Spek et al., 2017b). In addition, mice that lack D3 demonstrate impaired bacterial clearance following infection (Boelen et al., 2009). These data suggest that D3 is important for bacterial killing, a hallmark of neutrophil function. We hypothesized that D3 plays a role in neutrophil function during bacterial infection by actively regulating local TH bioavailability. Here we show that bacterial meningitis results in an altered TH profile at the site of infection, consistent with elevated D3 activity in infiltrating neutrophils. In addition, a lack of D3 results in impaired survival during bacterial meningitis in a zebrafish model. Finally, neutrophils derived from D3KO mice display functional abnormalities compared to wildtype cells. Our data suggest that neutrophil function during infection requires strict control of TH availability for which D3 appears essential.

Materials and Methods

Study Approval

All animal and human experiments were conducted in compliance with institutional and (inter)national guidelines and regulations. In the case of the Dutch Bacterial Meningitis Cohort, written informed consent was obtained from all participating patients or their legally authorized representatives prior to study inclusion (Bijlsma et al., 2016).

Human subjects

CSF samples were derived from the Dutch Bacterial Meningitis Cohort which includes adults with community-acquired bacterial meningitis with positive CSF cultures. Samples were obtained from the initial diagnostic lumbar puncture. These patients were identified by the Netherlands Reference

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74

Laboratory for Bacterial Meningitis as described in detail previously (Brouwer et al., 2010). The study protocol was approved by the Academic Medical Center Medical Ethical Committee and written informed consent was obtained from all participating patients or their legally authorized representatives prior to study inclusion. Control samples were derived from residual CSF obtained for diagnostic purposes from patients with thunderclap headache in whom a lumbar puncture was performed to rule out subarachnoid hemorrhage. Control CSF samples were used anonymously, in accordance with the Dutch Code of Conduct for the Secondary Use of Human Tissue (version 2011) issued by the Federation of Dutch Medical Scientific Societies. The full code of conduct is available online:

https://www.federa.org/sites/default/files/images/print_version_code_of_conduct_english.pdf

Thyroid hormone measurements in CSF

Thyroid hormone concentrations in CSF were measured using LC-MS/MS as described previously (Ackermans et al., 2012, de Vries et al., 2014a) with some minor modifications detailed below. Samples were deproteinized by adding 100 µl of acetonitrile to 30 µl of CSF together with 5 µl of 13C6-labeled internal standards. Following centrifugation, supernatants were transferred to glass tubes and dried using a SpeedVac. The residue was resuspended in 50 µl of 0.1% NH4OH and TH concentrations were measured on an Acquity UPLC- Xevo TQ-S tandem mass spectrometer system (Waters) as previously described for tissue (de Vries et al., 2014a, Ackermans et al., 2012). The detection limit of our assay was ± 0.1 nmol/L. Samples were measured in consecutive runs, the same control samples were included in all runs to detect inter-assay variation. Outcome was defined based on the Glasgow Outcome Scale (Jennett et al., 1976). Scoring is as follows: 1=death, 2=vegetative state, 3=severe disability, 4=moderate disability, and 5=mild or no disability. Unfavorable and favorable outcome were defined as a Glasgow Outcome Scale (GOS) of 1-4 and a GOS of 5 respectively.

Zebrafish embryo care and deiodinase knockdown

Zebrafish handling, embryo care and microinjections were performed as described previously (Jim et al., 2016). Embryos from the transparent casper zebrafish line (Tg(mitfaw2/w2;roya9/a9)) and the Tg(mpx:GFP)i114 line with GFP-labelled neutrophils were used. An antisense oligonucleotide morpholino (MO) knockdown approach was used to transiently block D3 expression as described previously (Heijlen et al., 2014). A standard control morpholino (SCMO) was used as a control. All MOs were purchased from Gene Tools. MO sequences were derived from Heijlen et al. (Heijlen et al., 2014) (D3MO against dio3b: 5’-CTGCGGAGCCCTGCAGCATCTCCAT-3’; SCMO: 5’-CCTCTTACCTCAGTTACAATTTATA-3’). The MO solutions were prepared in sterile distilled water and 0.5 % (w/v) phenol red solution (Sigma-Aldrich; P0290) to aid visualization of the injection process. The optimal concentration of 0.4 mM and injection volume 2 nl were used as previously described (Heijlen et al., 2014). The volume was injected into the yolk of 1- to 4-cell stage embryos, resulting in a delivery of approximately 6.6 ng MO per embryo. Embryos were raised at 28°C in E3 medium (5.0 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl·2H2O, 0.33 mM MgCl2·6H2O) supplemented with 0.3 mg/L methylene blue. The specificity of the D3MO used here has been previously demonstrated by rescue experiments using human D3 mRNA (Heijlen et al., 2014)

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5

and results in a decrease in D3 activity levels of 96%. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (zfin.org). All protocols adhered to the international guidelines specified by the European Council Directive 86/609/EEC and were conducted in compliance with institutional and national guidelines and regulations.

Bacterial strains and culture

Streptococcus pneumoniae D39 serotype 2 wild-type strain and a red fluorescent S. pneumoniae D39 HlpA-mCherry mutant strain were used (Avery et al., 1944, Beilharz et al., 2015). The bacterial culture method and injection process has been described in detail previously (Jim et al., 2016). Briefly, bacteria were grown overnight on Columbia agar plates supplemented with 5% defibrinated sheep blood in a humidified atmosphere with 5% CO2.Chloramphenicol (4,5 µg/ml) was added to agar plates for selection of the HlpA-mCherry mutant strain. Following overnight culture, bacteria were grown to mid log phase in Todd Hewitt broth with yeast extract, harvested by centrifugation, washed and suspended in sterile 0.5 % (w/v) phenol red solution (Sigma-Aldrich) to aid visualization of the injection process. The number of colony-forming units (CFU) per injection was determined by quantitative plating of the injection volume.

Infection of zebrafish embryos

D3MO and SCMO knockdown casper zebrafish embryos were infected with 500 CFU of wild-type S. pneumoniae D39 in the hindbrain ventricle as previously described, resulting in pneumococcal meningitis (Jim et al., 2016). The mortality rate was determined by monitoring live and dead embryos at fixed time points between 24 and 96 hours post infection (hpi). The experiment was repeated 3 times independently (total n=60 embryos/group).

Zebrafish embryo imaging and analysis

D3MO and SCMO knockdown Tg(mpx:GFP)i114 zebrafish embryos expressing green fluorescent neutrophils were injected with red fluorescent S. pneumoniae D39 HlpA-mCherry mutant and harvested at fixed time points between 1 and 5 hpi. Total neutrophil count was measured in D3MO and SCMO knockdown Tg(mpx:GFP)i114 zebrafish embryos at 2 days post fertilization. Embryos were fixed overnight in 4% paraformaldehyde in PBS. For optimal imaging, embryos were embedded in 1% low-melting-point agarose dissolved in PBS in an open uncoated 8-well microscopy μ-Slide (http://ibidi. com). Confocal images were generated with a Leica TCS SP8 Confocal Microscope and analyzed using Leica Application Suite X software and ImageJ.

D3KO mice care and procedures

D3KO mice in a CD-1 background harboring a previously described mutation were used (Hernandez et al., 2006). As D3KO mice are subfertile (Hernandez et al., 2006), D3KO animals were generated using heterozygous breeders. Wildtype (WT) littermates were used as controls. Both female and male WT and D3KO mice were used. Animals were housed under 12 hour light/12 hour dark cycles and ad libitum

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Laboratory for Bacterial Meningitis as described in detail previously (Brouwer et al., 2010). The study protocol was approved by the Academic Medical Center Medical Ethical Committee and written informed consent was obtained from all participating patients or their legally authorized representatives prior to study inclusion. Control samples were derived from residual CSF obtained for diagnostic purposes from patients with thunderclap headache in whom a lumbar puncture was performed to rule out subarachnoid hemorrhage. Control CSF samples were used anonymously, in accordance with the Dutch Code of Conduct for the Secondary Use of Human Tissue (version 2011) issued by the Federation of Dutch Medical Scientific Societies. The full code of conduct is available online:

https://www.federa.org/sites/default/files/images/print_version_code_of_conduct_english.pdf

Thyroid hormone measurements in CSF

Thyroid hormone concentrations in CSF were measured using LC-MS/MS as described previously (Ackermans et al., 2012, de Vries et al., 2014a) with some minor modifications detailed below. Samples were deproteinized by adding 100 µl of acetonitrile to 30 µl of CSF together with 5 µl of 13C6-labeled internal standards. Following centrifugation, supernatants were transferred to glass tubes and dried using a SpeedVac. The residue was resuspended in 50 µl of 0.1% NH4OH and TH concentrations were measured on an Acquity UPLC- Xevo TQ-S tandem mass spectrometer system (Waters) as previously described for tissue (de Vries et al., 2014a, Ackermans et al., 2012). The detection limit of our assay was ± 0.1 nmol/L. Samples were measured in consecutive runs, the same control samples were included in all runs to detect inter-assay variation. Outcome was defined based on the Glasgow Outcome Scale (Jennett et al., 1976). Scoring is as follows: 1=death, 2=vegetative state, 3=severe disability, 4=moderate disability, and 5=mild or no disability. Unfavorable and favorable outcome were defined as a Glasgow Outcome Scale (GOS) of 1-4 and a GOS of 5 respectively.

Zebrafish embryo care and deiodinase knockdown

Zebrafish handling, embryo care and microinjections were performed as described previously (Jim et al., 2016). Embryos from the transparent casper zebrafish line (Tg(mitfaw2/w2;roya9/a9)) and the Tg(mpx:GFP)i114 line with GFP-labelled neutrophils were used. An antisense oligonucleotide morpholino (MO) knockdown approach was used to transiently block D3 expression as described previously (Heijlen et al., 2014). A standard control morpholino (SCMO) was used as a control. All MOs were purchased from Gene Tools. MO sequences were derived from Heijlen et al. (Heijlen et al., 2014) (D3MO against dio3b: 5’-CTGCGGAGCCCTGCAGCATCTCCAT-3’; SCMO: 5’-CCTCTTACCTCAGTTACAATTTATA-3’). The MO solutions were prepared in sterile distilled water and 0.5 % (w/v) phenol red solution (Sigma-Aldrich; P0290) to aid visualization of the injection process. The optimal concentration of 0.4 mM and injection volume 2 nl were used as previously described (Heijlen et al., 2014). The volume was injected into the yolk of 1- to 4-cell stage embryos, resulting in a delivery of approximately 6.6 ng MO per embryo. Embryos were raised at 28°C in E3 medium (5.0 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl·2H2O, 0.33 mM MgCl2·6H2O) supplemented with 0.3 mg/L methylene blue. The specificity of the D3MO used here has been previously demonstrated by rescue experiments using human D3 mRNA (Heijlen et al., 2014)

75

5

and results in a decrease in D3 activity levels of 96%. Zebrafish were handled in compliance with the local animal welfare regulations and maintained according to standard protocols (zfin.org). All protocols adhered to the international guidelines specified by the European Council Directive 86/609/EEC and were conducted in compliance with institutional and national guidelines and regulations.

Bacterial strains and culture

Streptococcus pneumoniae D39 serotype 2 wild-type strain and a red fluorescent S. pneumoniae D39 HlpA-mCherry mutant strain were used (Avery et al., 1944, Beilharz et al., 2015). The bacterial culture method and injection process has been described in detail previously (Jim et al., 2016). Briefly, bacteria were grown overnight on Columbia agar plates supplemented with 5% defibrinated sheep blood in a humidified atmosphere with 5% CO2.Chloramphenicol (4,5 µg/ml) was added to agar plates for selection of the HlpA-mCherry mutant strain. Following overnight culture, bacteria were grown to mid log phase in Todd Hewitt broth with yeast extract, harvested by centrifugation, washed and suspended in sterile 0.5 % (w/v) phenol red solution (Sigma-Aldrich) to aid visualization of the injection process. The number of colony-forming units (CFU) per injection was determined by quantitative plating of the injection volume.

Infection of zebrafish embryos

D3MO and SCMO knockdown casper zebrafish embryos were infected with 500 CFU of wild-type S. pneumoniae D39 in the hindbrain ventricle as previously described, resulting in pneumococcal meningitis (Jim et al., 2016). The mortality rate was determined by monitoring live and dead embryos at fixed time points between 24 and 96 hours post infection (hpi). The experiment was repeated 3 times independently (total n=60 embryos/group).

Zebrafish embryo imaging and analysis

D3MO and SCMO knockdown Tg(mpx:GFP)i114 zebrafish embryos expressing green fluorescent neutrophils were injected with red fluorescent S. pneumoniae D39 HlpA-mCherry mutant and harvested at fixed time points between 1 and 5 hpi. Total neutrophil count was measured in D3MO and SCMO knockdown Tg(mpx:GFP)i114 zebrafish embryos at 2 days post fertilization. Embryos were fixed overnight in 4% paraformaldehyde in PBS. For optimal imaging, embryos were embedded in 1% low-melting-point agarose dissolved in PBS in an open uncoated 8-well microscopy μ-Slide (http://ibidi. com). Confocal images were generated with a Leica TCS SP8 Confocal Microscope and analyzed using Leica Application Suite X software and ImageJ.

D3KO mice care and procedures

D3KO mice in a CD-1 background harboring a previously described mutation were used (Hernandez et al., 2006). As D3KO mice are subfertile (Hernandez et al., 2006), D3KO animals were generated using heterozygous breeders. Wildtype (WT) littermates were used as controls. Both female and male WT and D3KO mice were used. Animals were housed under 12 hour light/12 hour dark cycles and ad libitum

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access to water and regular chow. Adult mice (3-5 months old) were sacrificed using CO2 asphyxiation. Bone marrow was isolated immediately from femurs and tibias and processed for flow cytometry staining or neutrophil isolation. Procedures were approved by the Institution of Animal Care and Use Committee at Maine Medical Center Research Institute.

Neutrophil isolation and culture

Neutrophils were isolated from whole bone marrow using the mouse neutrophil isolation kit (Miltenyi Biotec) according to manufacturer’s instructions. Following isolation, neutrophils were washed in PBS (room temperature), resuspended in HEPES medium (Boxio et al., 2004) supplemented with 1 mg/ ml glucose and immediately used for further experiments. Neutrophil purity was assessed using flow cytometry staining for the highly specific murine neutrophil marker Ly6G (Daley et al., 2008) (clone 1A8, BD Biosciences) and was always at least 90%.

Flow cytometric analysis of whole bone marrow

Whole bone marrow was stained using a panel of fluorescently labelled antibodies (listed in Table 5.1). All samples were incubated with mouse FC block (BD Biosciences) prior to staining and relevant isotype control antibodies were used to control for background staining. Bone marrow cell populations from both D3KO and WT mice were quantified using a MACSQuant flow cytometer (Miltenyi Biotec) and data was analyzed using FlowJo software (v.10). Flow cytometry gating of whole bone marrow is detailed in Supplemental Figure 5.1. Cells that expressed CD19, CD335 or CD3e were excluded from analysis (markers for B cells (Harwood and Batista, 2008), NK cells (Guerriero et al., 2011) and T cells (Leo et al., 1987) respectively). The following populations were identified: early hematopoietic blast cells CD117+/CD11b-; neutrophil precusors CD117+/CD11b+; monocyte precursors Ly-6G-/Ly-6C+/CD11blo; monocyte Ly-6G-/Ly-6C+/CD11bhi; neutrophil Ly-6G+/Ly-6C-/CD11bhi.

Protein target Fluorescent

conjugate Species raised in; monoclonal or polyclonal

Clone Catalog # Manufacturer

Ly-6G PerCP-Cy5.5 Rat IgG2A; monoclonal 1A8 560602 BD Biosciences Ly-6C PE Rat IgG2c; monoclonal HK1.4 12-5932 eBioscience CD117 (cKit) APC Rat IgG2b; monoclonal 2B8 17-1171 eBioscience CD11b APC-Cy7 Rat IgG2b; monoclonal M1/70 557657 BD Biosciences CD19 PE-Cy7 Rat IgG2A; monoclonal 1D3 25-0193 eBioscience CD335 PE-Cy7 Rat IgG2A; monoclonal 29A1.4 25-3351 eBioscience CD3e PE-Cy7 Armenian hamster;

monoclonal

145-2C11

25-0031 eBioscience CD16/CD32 (FC block) Rat IgG2A; monoclonal 93 14-0161 eBioscience

Table 5.1; Flow cytometry antibodies

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Unfavourable Favourable 0 2 4 6 8 CS Fr T3 nmol/L P=0.06 Unfavourable Favourable 0 10 20 30 40 CS FT 4n mo l/L P=0.32 Controls Patients 0 2 4 6 8 CS Fr T3 nmol/L P=<0.001 Controls Patients 0 10 20 30 40 CS FT 4n mo l/L P=<0.001

A

B

Figure 5.1 Bacterial meningitis changes thyroid hormone profile in CSF. (A) TH concentrations measured using LC/

MSMS in CSF samples from bacterial meningitis patients (n=93) and controls with benign thunderclap headache (n=9). T3 and T2 were undetectable in all samples (<± 0.1 nmol/L) (B) Comparison of CSF T4 and rT3 levels in CSF from patients with meningitis with unfavorable outcome (n=27) and patients with favorable outcome (n=48). No outcome data were available for 18 patients. Data are presented as median ±IQR. P-values for Mann Whitney-U test are indicated.

Analysis of neutrophil counts in whole blood

After euthanasia, blood was extracted from the cava vein of D3KO and WT mice and 40 µl of each sample was collected in EDTA microtainer tubes. Samples were placed on ice and used no later than 4 h after extraction. When ready for analysis, samples were mixed 10 times by inversion and analyzed automatically using a ProCyte DX Hematology Analyzer (IDEXX Laboratories).

Neutrophil apoptosis

Freshly isolated neutrophils (2x105/ml) were incubated under end-over-end rotation at 37°C. Samples were harvested at fixed timepoints and stained for Annexin-V and propidium iodide (PI) using the Annexin-V apoptosis detection kit (eBioscience). Samples were run on a MACSQuant flow cytometer (Miltenyi Biotec) and data was analyzed using FlowJo software (v.10).

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access to water and regular chow. Adult mice (3-5 months old) were sacrificed using CO2 asphyxiation. Bone marrow was isolated immediately from femurs and tibias and processed for flow cytometry staining or neutrophil isolation. Procedures were approved by the Institution of Animal Care and Use Committee at Maine Medical Center Research Institute.

Neutrophil isolation and culture

Neutrophils were isolated from whole bone marrow using the mouse neutrophil isolation kit (Miltenyi Biotec) according to manufacturer’s instructions. Following isolation, neutrophils were washed in PBS (room temperature), resuspended in HEPES medium (Boxio et al., 2004) supplemented with 1 mg/ ml glucose and immediately used for further experiments. Neutrophil purity was assessed using flow cytometry staining for the highly specific murine neutrophil marker Ly6G (Daley et al., 2008) (clone 1A8, BD Biosciences) and was always at least 90%.

Flow cytometric analysis of whole bone marrow

Whole bone marrow was stained using a panel of fluorescently labelled antibodies (listed in Table 5.1). All samples were incubated with mouse FC block (BD Biosciences) prior to staining and relevant isotype control antibodies were used to control for background staining. Bone marrow cell populations from both D3KO and WT mice were quantified using a MACSQuant flow cytometer (Miltenyi Biotec) and data was analyzed using FlowJo software (v.10). Flow cytometry gating of whole bone marrow is detailed in Supplemental Figure 5.1. Cells that expressed CD19, CD335 or CD3e were excluded from analysis (markers for B cells (Harwood and Batista, 2008), NK cells (Guerriero et al., 2011) and T cells (Leo et al., 1987) respectively). The following populations were identified: early hematopoietic blast cells CD117+/CD11b-; neutrophil precusors CD117+/CD11b+; monocyte precursors Ly-6G-/Ly-6C+/CD11blo; monocyte Ly-6G-/Ly-6C+/CD11bhi; neutrophil Ly-6G+/Ly-6C-/CD11bhi.

Protein target Fluorescent

conjugate Species raised in; monoclonal or polyclonal

Clone Catalog # Manufacturer

Ly-6G PerCP-Cy5.5 Rat IgG2A; monoclonal 1A8 560602 BD Biosciences Ly-6C PE Rat IgG2c; monoclonal HK1.4 12-5932 eBioscience CD117 (cKit) APC Rat IgG2b; monoclonal 2B8 17-1171 eBioscience CD11b APC-Cy7 Rat IgG2b; monoclonal M1/70 557657 BD Biosciences CD19 PE-Cy7 Rat IgG2A; monoclonal 1D3 25-0193 eBioscience CD335 PE-Cy7 Rat IgG2A; monoclonal 29A1.4 25-3351 eBioscience CD3e PE-Cy7 Armenian hamster;

monoclonal

145-2C11

25-0031 eBioscience CD16/CD32 (FC block) Rat IgG2A; monoclonal 93 14-0161 eBioscience

Table 5.1; Flow cytometry antibodies

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5

Unfavourable Favourable 0 2 4 6 8 CS Fr T3 nmol/L P=0.06 Unfavourable Favourable 0 10 20 30 40 CS FT 4n mo l/L P=0.32 Controls Patients 0 2 4 6 8 CS Fr T3 nmol/L P=<0.001 Controls Patients 0 10 20 30 40 CS FT 4n mo l/L P=<0.001

A

B

Figure 5.1 Bacterial meningitis changes thyroid hormone profile in CSF. (A) TH concentrations measured using LC/

MSMS in CSF samples from bacterial meningitis patients (n=93) and controls with benign thunderclap headache (n=9). T3 and T2 were undetectable in all samples (<± 0.1 nmol/L) (B) Comparison of CSF T4 and rT3 levels in CSF from patients with meningitis with unfavorable outcome (n=27) and patients with favorable outcome (n=48). No outcome data were available for 18 patients. Data are presented as median ±IQR. P-values for Mann Whitney-U test are indicated.

Analysis of neutrophil counts in whole blood

After euthanasia, blood was extracted from the cava vein of D3KO and WT mice and 40 µl of each sample was collected in EDTA microtainer tubes. Samples were placed on ice and used no later than 4 h after extraction. When ready for analysis, samples were mixed 10 times by inversion and analyzed automatically using a ProCyte DX Hematology Analyzer (IDEXX Laboratories).

Neutrophil apoptosis

Freshly isolated neutrophils (2x105/ml) were incubated under end-over-end rotation at 37°C. Samples were harvested at fixed timepoints and stained for Annexin-V and propidium iodide (PI) using the Annexin-V apoptosis detection kit (eBioscience). Samples were run on a MACSQuant flow cytometer (Miltenyi Biotec) and data was analyzed using FlowJo software (v.10).

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Neutrophil phagocytosis

Zymosan bioparticles fluorescently labelled with the pH sensitive fluorophore pHrodo green (Molecular Probes) were opsonized using opsonizing reagent (Molecular Probes) according to manufacturer’s instructions. Zymosan is a yeast particle and phagocytic stimulus. Neutrophils were incubated with opsonized pHrodo-conjugated zymosan (MOI 5) at 37°C or on ice (as a control) for 1 hour. Fluorescence was quantified using a MACSQuant flow cytometer (Miltenyi Biotec) and FlowJo software (v.10). Flow cytometry gating of phagocytosing neutrophils is detailed in Supplemental Figure 5.2. Neutrophils in the FITC+ gate were considered phagocytosing neutrophils.

Neutrophil NAPDH oxidase activity

Extracellular H2O2 production was determined as a measure for NADPH oxidase activity using the Amplex Red Hydrogen Peroxide Kit (Molecular Probes). Neutrophils (2x104) were incubated with PMA (100ng/ml) in the presence of Amplex Red (50 µM) and HRP (0.1 U/ml). Fluorescence (excitation 535 nm; emission 595 nm) was measured at 2 minute intervals for 30 minutes using a Gen5 2.09 microplate reader (BioTek).

Statistics

Statistical tests used are described in detail in the relevant figure legends. P-values <0.05 were considered statistically significant. Tests were performed using Graphpad Prism 7 or SPSS v. 23.

Results

Bacterial meningitis alters thyroid hormone concentrations in the cerebrospinal fluid

To study whether infiltrating neutrophils can alter TH concentrations at the site of infection we measured TH concentrations in cerebrospinal fluid (CSF) from the diagnostic lumbar puncture of patients with bacterial meningitis (n=95) versus controls. CSF samples from patients with benign thunderclap headache (n=9) who had undergone a diagnostic lumbar puncture to rule out subarachnoid hemorrhage were used as a control. Bacterial meningitis resulted in profound changes in CSF TH concentrations. Both T4 and rT3 were strongly elevated in CSF from bacterial meningitis patients compared to controls (Figure 5.1A). T3, 3,5-T2 and 3,3’-T2 were undetectable in all samples (detection limit ± 0.1 nmol/L). In addition, we analysed whether CSF TH concentrations were correlated with clinical outcome. No statistically significant differences in CSF TH concentrations were found between bacterial meningitis patients with an unfavourable or a favourable outcome, as defined on the Glasgow Outcome Score (Figure 5.1B).

The median value of CSF T4 was 6.04 nmol/L (IQR 2.31-12.81) in bacterial meningitis patients, significantly higher than the 0.92 nmol/L (IQR 0.44-1.16) found in CSF from controls. CSF rT3 concentrations were also higher during bacterial meningitis (median 1.01 nmol/L; IQR 0.38-1.84) compared to controls (median 0.07 nmol/L; IQR 0.055-0.12).

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0 24 48 72 96 0 25 50 75 100 hpi Percent su rv ival SCMO D3MO P<0.001 1 3 5 0 20 40 60

Hindbrain ventricle neutrophil count

hpi SCMO D3MO P=0.006 _**

A

C

PMNSpneu

SCMO 5 hpi D3MO 5 hpi

Neutrophils/embryo

Zebrafish embryo survival curve

B

SCMO D3MO 0 50 100 150 200

Baseline whole embryo Neutrophil count

Neutrophils/embry

o

Figure 5.2 Effect of D3 knockdown during pneumococcal meningitis in zebrafish embryos. (A) Survival curve for

zebrafish embryos treated with standard control morpholino (SCMO; n=60) or D3 morpholino (D3MO; n=60) and infected with approximately 500 CFU of wildtype S.pneumoniae. Pooled data from 3 independent experiments is shown. Error bars represent SE. P-value for log-rank (Mantel-Cox) test is indicated. Hpi = hours post infection. (B) Quantification of whole body baseline neutrophils in uninfected D3MO and SCMO Tg(mpx:GFP) zebrafish embryos with fluorescent neutrophils from confocal microscopy images (C) Confocal microscopy images of S.pneumoniae D39 HlpA-mCherry infected D3MO and SCMO Tg(mpx:GFP) zebrafish embryos with fluorescent neutrophils (green) and pneumococci (red). Scale bar= 100 µm. Quantification of neutrophils in the hindbrain ventricle from confocal microscopy images (n=10-12 embryos/group). Data represent mean ±SEM. P-value for two-way ANOVA is indicated. Post-hoc analysis (Bonferroni) p-value: **p<0.01

D3 knockdown impairs zebrafish embryo survival and neutrophil migration during bacterial meningitis

As infiltrating neutrophils appear to affect TH concentrations at the site of infection, we next assessed whether cellular TH bioavailability could also affect neutrophil function in vivo. We combined a validated model for knockdown of D3 in zebrafish embryos using morpholino oligonucleotide gene knockdown technology (Heijlen et al., 2014) with an established zebrafish embryo model for pneumococcal meningitis (Jim et al., 2016) and determined the effect ofmodulating TH bioavailability during bacterial meningitis. Following treatment with a morpholino targeting D3 (D3MO) or a standard

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Neutrophil phagocytosis

Zymosan bioparticles fluorescently labelled with the pH sensitive fluorophore pHrodo green (Molecular Probes) were opsonized using opsonizing reagent (Molecular Probes) according to manufacturer’s instructions. Zymosan is a yeast particle and phagocytic stimulus. Neutrophils were incubated with opsonized pHrodo-conjugated zymosan (MOI 5) at 37°C or on ice (as a control) for 1 hour. Fluorescence was quantified using a MACSQuant flow cytometer (Miltenyi Biotec) and FlowJo software (v.10). Flow cytometry gating of phagocytosing neutrophils is detailed in Supplemental Figure 5.2. Neutrophils in the FITC+ gate were considered phagocytosing neutrophils.

Neutrophil NAPDH oxidase activity

Extracellular H2O2 production was determined as a measure for NADPH oxidase activity using the Amplex Red Hydrogen Peroxide Kit (Molecular Probes). Neutrophils (2x104) were incubated with PMA (100ng/ml) in the presence of Amplex Red (50 µM) and HRP (0.1 U/ml). Fluorescence (excitation 535 nm; emission 595 nm) was measured at 2 minute intervals for 30 minutes using a Gen5 2.09 microplate reader (BioTek).

Statistics

Statistical tests used are described in detail in the relevant figure legends. P-values <0.05 were considered statistically significant. Tests were performed using Graphpad Prism 7 or SPSS v. 23.

Results

Bacterial meningitis alters thyroid hormone concentrations in the cerebrospinal fluid

To study whether infiltrating neutrophils can alter TH concentrations at the site of infection we measured TH concentrations in cerebrospinal fluid (CSF) from the diagnostic lumbar puncture of patients with bacterial meningitis (n=95) versus controls. CSF samples from patients with benign thunderclap headache (n=9) who had undergone a diagnostic lumbar puncture to rule out subarachnoid hemorrhage were used as a control. Bacterial meningitis resulted in profound changes in CSF TH concentrations. Both T4 and rT3 were strongly elevated in CSF from bacterial meningitis patients compared to controls (Figure 5.1A). T3, 3,5-T2 and 3,3’-T2 were undetectable in all samples (detection limit ± 0.1 nmol/L). In addition, we analysed whether CSF TH concentrations were correlated with clinical outcome. No statistically significant differences in CSF TH concentrations were found between bacterial meningitis patients with an unfavourable or a favourable outcome, as defined on the Glasgow Outcome Score (Figure 5.1B).

The median value of CSF T4 was 6.04 nmol/L (IQR 2.31-12.81) in bacterial meningitis patients, significantly higher than the 0.92 nmol/L (IQR 0.44-1.16) found in CSF from controls. CSF rT3 concentrations were also higher during bacterial meningitis (median 1.01 nmol/L; IQR 0.38-1.84) compared to controls (median 0.07 nmol/L; IQR 0.055-0.12).

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5

0 24 48 72 96 0 25 50 75 100 hpi Percent su rv ival SCMO D3MO P<0.001 1 3 5 0 20 40 60

Hindbrain ventricle neutrophil count

hpi SCMO D3MO P=0.006 _**

A

C

PMNSpneu

SCMO 5 hpi D3MO 5 hpi

Neutrophils/embryo

Zebrafish embryo survival curve

B

SCMO D3MO 0 50 100 150 200

Baseline whole embryo Neutrophil count

Neutrophils/embry

o

Figure 5.2 Effect of D3 knockdown during pneumococcal meningitis in zebrafish embryos. (A) Survival curve for

zebrafish embryos treated with standard control morpholino (SCMO; n=60) or D3 morpholino (D3MO; n=60) and infected with approximately 500 CFU of wildtype S.pneumoniae. Pooled data from 3 independent experiments is shown. Error bars represent SE. P-value for log-rank (Mantel-Cox) test is indicated. Hpi = hours post infection. (B) Quantification of whole body baseline neutrophils in uninfected D3MO and SCMO Tg(mpx:GFP) zebrafish embryos with fluorescent neutrophils from confocal microscopy images (C) Confocal microscopy images of S.pneumoniae D39 HlpA-mCherry infected D3MO and SCMO Tg(mpx:GFP) zebrafish embryos with fluorescent neutrophils (green) and pneumococci (red). Scale bar= 100 µm. Quantification of neutrophils in the hindbrain ventricle from confocal microscopy images (n=10-12 embryos/group). Data represent mean ±SEM. P-value for two-way ANOVA is indicated. Post-hoc analysis (Bonferroni) p-value: **p<0.01

D3 knockdown impairs zebrafish embryo survival and neutrophil migration during bacterial meningitis

As infiltrating neutrophils appear to affect TH concentrations at the site of infection, we next assessed whether cellular TH bioavailability could also affect neutrophil function in vivo. We combined a validated model for knockdown of D3 in zebrafish embryos using morpholino oligonucleotide gene knockdown technology (Heijlen et al., 2014) with an established zebrafish embryo model for pneumococcal meningitis (Jim et al., 2016) and determined the effect ofmodulating TH bioavailability during bacterial meningitis. Following treatment with a morpholino targeting D3 (D3MO) or a standard

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control morpholino (SCMO), zebrafish embryos were injected with S. pneumoniae D39, serotype 2, in the hindbrain ventricle. This injection leads to meningitis (Jim et al., 2016). In the D3MO zebrafish embryos, survival of pneumococcal meningitis was significantly decreased (Figure 5.2A). In addition, although the whole body neutrophil count at baseline did not differ between the groups (Figure 5.2B), the amount of infiltrating neutrophils at the site of infection was lower in D3MO zebrafish embryos as compared to controls (Figure 5.2C).

D3KO neutrophils exhibit impaired NADPH oxidase activity

To determine whether D3 does indeed play a functional role in neutrophils we assessed neutrophil function in D3KO mice and their WT littermates. Impaired neutrophil function could be due to reduced availability of neutrophils caused by a defect in neutrophil production by the bone marrow. Therefore, we studied the hematopoietic populations present in the bone marrow of D3KO and WT mice. There were no differences in relative amounts of bone marrow neutrophils between D3KO and WT mice, nor in amounts of other cell types of the hematopoietic lineage (Figure 5.3A). The percentage of circulating neutrophils in venous blood was also unchanged in D3KO mice (Figure 5.3A).

To determine whether D3 directly affects neutrophil function, we isolated neutrophils from the bone marrow of D3KO and WT mice and assessed neutrophil survival, phagocytosis of zymosan particles and NADPH oxidase activity ex vivo. Neutrophil survival and phagocytosis were both unchanged in D3KO mice (Figure 5.3B-C). In contrast NADPH oxidase activity, as measured by the cells ability to produce H2O2 upon stimulation, was impaired in D3KO neutrophils (Figure 5.3D). Interestingly, the reduction in NADPH oxidase activity was due to the effect in female animals (Figure 5.3D). Following this finding we re-analyzed the hematopoietic bone marrow and neutrophil phagocytosis data taking into account the gender of the animals. No difference was found between D3KO and WT animals when only comparing animals of the same gender. Neutrophil survival could not be re-analyzed as there were insufficient female samples to analyze the data separately for both genders.

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0 10 20 30 1¥105 3¥105 5¥105 NADPH oxidase activity males Time (mins) RF U P=n.s. 0 WT D3KO Neutrophils in venous blood Male W T Male D 3KO Female WT Female D3KO 0 20 40 60 80 %o ft otal ce lls P=n.s. HSC PMN

prec Mono prec Mono PMN 0 1 2 3 4 10 15 20 25

Bone marrow populations

%o ft otal ce lls WTD3KO P=n.s. 0 2 4 6 8 0 25 50 75 100 24 Apoptosis D3KO healthy WT apoptotic D3KO apoptotic Hours %o ft otal ce lls WT healthy P=n.s. Neutrophil phagocytosis WT

ice D3KO ice WT D3KO 0.0 0.5 1.0 1.5 2.0 Zy mo sa np Hrod o( MF I) % Phagocytosing neutrophils WT D3KO 0 10 20 30 40 50 %o ft ot al neutrophils 0 10 20 30 0 2¥105 4¥105 6¥105 NADPH oxidase activity females Time (mins) RF U P=<0.001 ****** ****** WT D3KO

A

B

C

D

WT D3KO 0 2¥106 4¥106 6¥106 8¥106 AUC females AU C * WT D3KO 0 2¥106 4¥106 6¥106 AUC males AU C

Figure 5.3 Neutrophil function in D3KO mice. (A) Hematopoietic bone marrow populations in D3KO mice (n=6) and WT

littermates (n=7) quantified using flow cytometry. Percentage of total cells is shown for early hematopoietic blast cells (HSC), neutrophil precursors (PMN prec), monocyte precursors (mono prec), monocytes (mono) and neutrophils (PMN). See Supplemental Figure 5.1 for gating. Neutrophils were quantified in venous blood derived from the cava vein of D3KO (n=6 male, n=5 female) and WT (n=7 male, n=3 female) mice. (B) Ex vivo survival of neutrophils incubated at 37°C. Samples were stained for Annexin V and propidium iodide (PI). Percentage of healthy cells (Annexin V-/PI-) and apoptotic cells (Annexin V+) are shown (n=3-4 mice per genotype per time point). (C) Neutrophils (D3KO n=7; WT n=5) were incubated with zymosan fluorescently labelled with pHrodo green (MOI 5) for 1 hour at 37°C or on ice. pHrodo+ cells (see Supplemental Figure 5.2 for gating) were considered phagocytosing neutrophils. MFI=median fluorescence intensity (D) D3KO and WT neutrophils were incubated with PMA. Fluorescence, indicating H2O2 release, was measured. The effect of D3KO on NADPH oxidase activity was only present in cells from female mice (D3KO n=5; WT n=3) and was not observed in cells from male mice (D3KO n=5, WT n=4). AUC was analyzed using unpaired two-tailed Student’s t-test *p<0.05. RFU=relative fluorescent units. (A-D) All data represent mean ±SEM. N-values indicate number of animals used. Assays were performed in triplicate. Experiments were repeated independently 2-4 times. P-values for two-way ANOVA are indicated. Post-hoc analysis (Bonferroni) p-values: *p<0.05 **p<0.01 ***p<0.001.

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control morpholino (SCMO), zebrafish embryos were injected with S. pneumoniae D39, serotype 2, in the hindbrain ventricle. This injection leads to meningitis (Jim et al., 2016). In the D3MO zebrafish embryos, survival of pneumococcal meningitis was significantly decreased (Figure 5.2A). In addition, although the whole body neutrophil count at baseline did not differ between the groups (Figure 5.2B), the amount of infiltrating neutrophils at the site of infection was lower in D3MO zebrafish embryos as compared to controls (Figure 5.2C).

D3KO neutrophils exhibit impaired NADPH oxidase activity

To determine whether D3 does indeed play a functional role in neutrophils we assessed neutrophil function in D3KO mice and their WT littermates. Impaired neutrophil function could be due to reduced availability of neutrophils caused by a defect in neutrophil production by the bone marrow. Therefore, we studied the hematopoietic populations present in the bone marrow of D3KO and WT mice. There were no differences in relative amounts of bone marrow neutrophils between D3KO and WT mice, nor in amounts of other cell types of the hematopoietic lineage (Figure 5.3A). The percentage of circulating neutrophils in venous blood was also unchanged in D3KO mice (Figure 5.3A).

To determine whether D3 directly affects neutrophil function, we isolated neutrophils from the bone marrow of D3KO and WT mice and assessed neutrophil survival, phagocytosis of zymosan particles and NADPH oxidase activity ex vivo. Neutrophil survival and phagocytosis were both unchanged in D3KO mice (Figure 5.3B-C). In contrast NADPH oxidase activity, as measured by the cells ability to produce H2O2 upon stimulation, was impaired in D3KO neutrophils (Figure 5.3D). Interestingly, the reduction in NADPH oxidase activity was due to the effect in female animals (Figure 5.3D). Following this finding we re-analyzed the hematopoietic bone marrow and neutrophil phagocytosis data taking into account the gender of the animals. No difference was found between D3KO and WT animals when only comparing animals of the same gender. Neutrophil survival could not be re-analyzed as there were insufficient female samples to analyze the data separately for both genders.

81

5

0 10 20 30 1¥105 3¥105 5¥105 NADPH oxidase activity males Time (mins) RF U P=n.s. 0 WT D3KO Neutrophils in venous blood Male W T Male D 3KO Female WT Female D3KO 0 20 40 60 80 %o ft otal ce lls P=n.s. HSC PMN

prec Mono prec Mono PMN 0 1 2 3 4 10 15 20 25

Bone marrow populations

%o ft otal ce lls WTD3KO P=n.s. 0 2 4 6 8 0 25 50 75 100 24 Apoptosis D3KO healthy WT apoptotic D3KO apoptotic Hours %o ft otal ce lls WT healthy P=n.s. Neutrophil phagocytosis WT

ice D3KO ice WT D3KO 0.0 0.5 1.0 1.5 2.0 Zy mo sa np Hrod o( MF I) % Phagocytosing neutrophils WT D3KO 0 10 20 30 40 50 %o ft ot al neutrophils 0 10 20 30 0 2¥105 4¥105 6¥105 NADPH oxidase activity females Time (mins) RF U P=<0.001 ****** ****** WT D3KO

A

B

C

D

WT D3KO 0 2¥106 4¥106 6¥106 8¥106 AUC females AU C * WT D3KO 0 2¥106 4¥106 6¥106 AUC males AU C

Figure 5.3 Neutrophil function in D3KO mice. (A) Hematopoietic bone marrow populations in D3KO mice (n=6) and WT

littermates (n=7) quantified using flow cytometry. Percentage of total cells is shown for early hematopoietic blast cells (HSC), neutrophil precursors (PMN prec), monocyte precursors (mono prec), monocytes (mono) and neutrophils (PMN). See Supplemental Figure 5.1 for gating. Neutrophils were quantified in venous blood derived from the cava vein of D3KO (n=6 male, n=5 female) and WT (n=7 male, n=3 female) mice. (B) Ex vivo survival of neutrophils incubated at 37°C. Samples were stained for Annexin V and propidium iodide (PI). Percentage of healthy cells (Annexin V-/PI-) and apoptotic cells (Annexin V+) are shown (n=3-4 mice per genotype per time point). (C) Neutrophils (D3KO n=7; WT n=5) were incubated with zymosan fluorescently labelled with pHrodo green (MOI 5) for 1 hour at 37°C or on ice. pHrodo+ cells (see Supplemental Figure 5.2 for gating) were considered phagocytosing neutrophils. MFI=median fluorescence intensity (D) D3KO and WT neutrophils were incubated with PMA. Fluorescence, indicating H2O2 release, was measured. The effect of D3KO on NADPH oxidase activity was only present in cells from female mice (D3KO n=5; WT n=3) and was not observed in cells from male mice (D3KO n=5, WT n=4). AUC was analyzed using unpaired two-tailed Student’s t-test *p<0.05. RFU=relative fluorescent units. (A-D) All data represent mean ±SEM. N-values indicate number of animals used. Assays were performed in triplicate. Experiments were repeated independently 2-4 times. P-values for two-way ANOVA are indicated. Post-hoc analysis (Bonferroni) p-values: *p<0.05 **p<0.01 ***p<0.001.

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Discussion

There is increasing evidence that innate immune cells are important TH target cells (van der Spek et al., 2017b). Neutrophils are known to contain the TH inactivating enzyme D3 (van der Spek et al., 2016, Boelen et al., 2008, Boelen et al., 2005). As mice that lack D3 exhibit impaired bacterial killing capacity (Boelen et al., 2009), D3 was hypothesized to play an important role in these cells. The present study is the first to demonstrate a functional role for D3 in neutrophils. Additionally, our data suggests that D3, and thus adequate regulation of intracellular TH levels, plays a crucial role in neutrophil function during infection in vivo.

Bacterial meningitis results in profound changes in CSF TH concentrations. Bacterial meningitis is a severe infectious disease of the central nervous system which is associated with substantial morbidity and mortality (Bijlsma et al., 2016, van de Beek et al., 2004) and characterized by high neutrophil infiltration into the CSF (Mook-Kanamori et al., 2011). The most common causative pathogen of bacterial meningitis is Streptococcus pneumoniae (70% of cases) (Bijlsma et al., 2016).

Bacterial meningitis patients had significantly higher levels of T4 in their CSF. As T4 is not generated locally but produced by the thyroid exclusively, the T4 observed in the CSF during bacterial meningitis must originate from the systemic circulation. This could be due to increased permeability of the blood-brain barrier, which is commonly observed during bacterial meningitis (Mook-Kanamori et al., 2011). Furthermore, T4 is known to concentrate at a localized site of infection as seen in a pulmonary abscess (Adelberg et al., 1971). During illness, serum TH levels undergo profound changes characterized by decreased serum T3 and T4 concentrations and increased rT3 concentrations (Fliers et al., 2015). This is known as nonthyroidal illness syndrome (NTIS) and is correlated with illness severity and outcome in a wide range of diseases (Fliers et al., 2015). During NTIS, serum T4 concentrations can drop as low as 40-45 nmol/L (Peeters et al., 2003) (reference range in our laboratory: 70–150 nmol/L). The concentration of CSF T4 is approximately 2.5% of serum T4 in healthy euthyroid humans (Hagen and Elliott, 1973). Assuming that all CSF T4 is derived from the circulation, this would mean that during bacterial meningitis at least 15% of the serum T4 concentration leaks to the CSF compartment. Higher rT3 concentrations were also observed in the CSF of bacterial meningitis patients compared to controls. Even though serum rT3 concentrations are expected to increase during bacterial meningitis, the concentrations we find in the CSF are too high to be explained by leakage from the circulation. Serum rT3 values can increase up to approximately 2 nmol/L during NTIS (Peeters et al., 2003, Boelen et al., 1993) (reference range in our laboratory 0.11-0.44 nmol/L). The median CSF rT3 concentration found in bacterial meningitis patients is thus approximately 50% of the serum rT3 concentrations found during NTIS. As this is much higher than the 15% of serum T4 found in the CSF, this cannot be explained by leakage from the circulation but is most likely the result of conversion of T4 by D3 within the CSF. A study in nonhuman primates demonstrated that the mean increase in CSF rT3 and CSF T4 after i.v. injection of the respective hormones was similar, or even slightly lower for rT3, indicating that the transport from the serum to the CSF compartment is normally comparable for T4 and rT3 (Chernow

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et al., 1983). Neutrophils contain significant amounts of D3 (van der Spek et al., 2016) and bacterial meningitis is characterized by large scale neutrophil infiltration into the CSF (Mook-Kanamori et al., 2011). Therefore, we conclude that changes in the TH profile of CSF during bacterial meningitis are very likely explained by leakage of T4 from the circulation due to increased blood-brain barrier permeability followed by local inactivation of T4 to rT3 by D3 in infiltrating neutrophils. These results suggest that infiltrating neutrophils are capable of altering TH concentrations at the site of infection.

D3 knockdown in zebrafish embryos results in impaired survival during pneumococcal meningitis as well as in reduced numbers of neutrophils at the site of infection. These results suggest that D3 is essential for the immune response against bacterial infection in vivo. This is in accordance with previous data in D3KO mice showing increased bacterial load after pulmonary infection with S.pneumoniae compared to wildtype (WT) mice (Boelen et al., 2009). The effects we observed in zebrafish are likely to be caused by impaired neutrophil function due to changes in intracellular TH concentrations in these important TH target cells. Although baseline whole body neutrophil count is not affected by D3 knockdown, D3MO zebrafish exhibit less infiltrating neutrophils at the site of infection. Mobilization of neutrophils is regulated by these cells’ ability to sense inflammatory mediators that guide them towards the site of infection in a process known as chemotaxis (Stephens et al., 2008). A possible mechanism could be via rT3, which initiates actin polymerization in astrocytes (Cheng et al., 2010). Therefore, a lack of D3 could impair neutrophil actin polymerization by decreasing rT3 concentrations, thereby reducing cellular mobility. It should be noted that there is no data available on the proposed effects of rT3 on neutrophil mobility, therefore this mechanism remains speculative. As D3 knockdown in zebrafish is known to affect development, we cannot exclude that the changes in survival are (partially) due to defects at the whole organism level due to the lack of D3 (Heijlen et al., 2014). However, the effects of D3MO on zebrafish survival are much less severe than the differences observed here (Heijlen et al., 2014). Neutrophils can also secrete these inflammatory mediators, thereby recruiting more cells from the circulation. Elevated T3 due to deficient D3 function could lead to abnormal production of chemokines needed for neutrophil recruitment.

Neutrophils have a variety of killing mechanisms at their disposal. Two of the main neutrophil antimicrobial mechanisms are phagocytosis followed by the release of reactive oxygen species (ROS) and antimicrobial proteins into the phagosome (Kolaczkowska and Kubes, 2013). ROS are produced by NADPH oxidase within the cell upon contact with a pathogen (Kolaczkowska and Kubes, 2013). Impaired neutrophil NAPDH oxidase activity in humans causes a syndrome known as chronic granulomatous disease which is characterized by recurrent severe bacterial infections due to impaired neutrophil killing (Dinauer, 2014). Primary murine D3KO neutrophils from female mice exhibit impaired NADPH oxidase activity. Interestingly, the reduction in NADPH oxidase activity was not observed in male animals. Sex differences in the immune response have been broadly described with in general, the female immune system exhibiting a greater inflammatory response than the male (Klein and Flanagan, 2016). This is confirmed by the increased NADPH oxidase response observed in neutrophils from female WT animals versus males. Following this finding in D3KO mice we re-analyzed the CSF TH concentrations from

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Discussion

There is increasing evidence that innate immune cells are important TH target cells (van der Spek et al., 2017b). Neutrophils are known to contain the TH inactivating enzyme D3 (van der Spek et al., 2016, Boelen et al., 2008, Boelen et al., 2005). As mice that lack D3 exhibit impaired bacterial killing capacity (Boelen et al., 2009), D3 was hypothesized to play an important role in these cells. The present study is the first to demonstrate a functional role for D3 in neutrophils. Additionally, our data suggests that D3, and thus adequate regulation of intracellular TH levels, plays a crucial role in neutrophil function during infection in vivo.

Bacterial meningitis results in profound changes in CSF TH concentrations. Bacterial meningitis is a severe infectious disease of the central nervous system which is associated with substantial morbidity and mortality (Bijlsma et al., 2016, van de Beek et al., 2004) and characterized by high neutrophil infiltration into the CSF (Mook-Kanamori et al., 2011). The most common causative pathogen of bacterial meningitis is Streptococcus pneumoniae (70% of cases) (Bijlsma et al., 2016).

Bacterial meningitis patients had significantly higher levels of T4 in their CSF. As T4 is not generated locally but produced by the thyroid exclusively, the T4 observed in the CSF during bacterial meningitis must originate from the systemic circulation. This could be due to increased permeability of the blood-brain barrier, which is commonly observed during bacterial meningitis (Mook-Kanamori et al., 2011). Furthermore, T4 is known to concentrate at a localized site of infection as seen in a pulmonary abscess (Adelberg et al., 1971). During illness, serum TH levels undergo profound changes characterized by decreased serum T3 and T4 concentrations and increased rT3 concentrations (Fliers et al., 2015). This is known as nonthyroidal illness syndrome (NTIS) and is correlated with illness severity and outcome in a wide range of diseases (Fliers et al., 2015). During NTIS, serum T4 concentrations can drop as low as 40-45 nmol/L (Peeters et al., 2003) (reference range in our laboratory: 70–150 nmol/L). The concentration of CSF T4 is approximately 2.5% of serum T4 in healthy euthyroid humans (Hagen and Elliott, 1973). Assuming that all CSF T4 is derived from the circulation, this would mean that during bacterial meningitis at least 15% of the serum T4 concentration leaks to the CSF compartment. Higher rT3 concentrations were also observed in the CSF of bacterial meningitis patients compared to controls. Even though serum rT3 concentrations are expected to increase during bacterial meningitis, the concentrations we find in the CSF are too high to be explained by leakage from the circulation. Serum rT3 values can increase up to approximately 2 nmol/L during NTIS (Peeters et al., 2003, Boelen et al., 1993) (reference range in our laboratory 0.11-0.44 nmol/L). The median CSF rT3 concentration found in bacterial meningitis patients is thus approximately 50% of the serum rT3 concentrations found during NTIS. As this is much higher than the 15% of serum T4 found in the CSF, this cannot be explained by leakage from the circulation but is most likely the result of conversion of T4 by D3 within the CSF. A study in nonhuman primates demonstrated that the mean increase in CSF rT3 and CSF T4 after i.v. injection of the respective hormones was similar, or even slightly lower for rT3, indicating that the transport from the serum to the CSF compartment is normally comparable for T4 and rT3 (Chernow

83

5

et al., 1983). Neutrophils contain significant amounts of D3 (van der Spek et al., 2016) and bacterial meningitis is characterized by large scale neutrophil infiltration into the CSF (Mook-Kanamori et al., 2011). Therefore, we conclude that changes in the TH profile of CSF during bacterial meningitis are very likely explained by leakage of T4 from the circulation due to increased blood-brain barrier permeability followed by local inactivation of T4 to rT3 by D3 in infiltrating neutrophils. These results suggest that infiltrating neutrophils are capable of altering TH concentrations at the site of infection.

D3 knockdown in zebrafish embryos results in impaired survival during pneumococcal meningitis as well as in reduced numbers of neutrophils at the site of infection. These results suggest that D3 is essential for the immune response against bacterial infection in vivo. This is in accordance with previous data in D3KO mice showing increased bacterial load after pulmonary infection with S.pneumoniae compared to wildtype (WT) mice (Boelen et al., 2009). The effects we observed in zebrafish are likely to be caused by impaired neutrophil function due to changes in intracellular TH concentrations in these important TH target cells. Although baseline whole body neutrophil count is not affected by D3 knockdown, D3MO zebrafish exhibit less infiltrating neutrophils at the site of infection. Mobilization of neutrophils is regulated by these cells’ ability to sense inflammatory mediators that guide them towards the site of infection in a process known as chemotaxis (Stephens et al., 2008). A possible mechanism could be via rT3, which initiates actin polymerization in astrocytes (Cheng et al., 2010). Therefore, a lack of D3 could impair neutrophil actin polymerization by decreasing rT3 concentrations, thereby reducing cellular mobility. It should be noted that there is no data available on the proposed effects of rT3 on neutrophil mobility, therefore this mechanism remains speculative. As D3 knockdown in zebrafish is known to affect development, we cannot exclude that the changes in survival are (partially) due to defects at the whole organism level due to the lack of D3 (Heijlen et al., 2014). However, the effects of D3MO on zebrafish survival are much less severe than the differences observed here (Heijlen et al., 2014). Neutrophils can also secrete these inflammatory mediators, thereby recruiting more cells from the circulation. Elevated T3 due to deficient D3 function could lead to abnormal production of chemokines needed for neutrophil recruitment.

Neutrophils have a variety of killing mechanisms at their disposal. Two of the main neutrophil antimicrobial mechanisms are phagocytosis followed by the release of reactive oxygen species (ROS) and antimicrobial proteins into the phagosome (Kolaczkowska and Kubes, 2013). ROS are produced by NADPH oxidase within the cell upon contact with a pathogen (Kolaczkowska and Kubes, 2013). Impaired neutrophil NAPDH oxidase activity in humans causes a syndrome known as chronic granulomatous disease which is characterized by recurrent severe bacterial infections due to impaired neutrophil killing (Dinauer, 2014). Primary murine D3KO neutrophils from female mice exhibit impaired NADPH oxidase activity. Interestingly, the reduction in NADPH oxidase activity was not observed in male animals. Sex differences in the immune response have been broadly described with in general, the female immune system exhibiting a greater inflammatory response than the male (Klein and Flanagan, 2016). This is confirmed by the increased NADPH oxidase response observed in neutrophils from female WT animals versus males. Following this finding in D3KO mice we re-analyzed the CSF TH concentrations from

The role of intracellular thyroid hormone metabolism in innate immune cells - Chapter 5: The thyroid hormone inactivating type 3 deiodinase is essential for optimal neutrophil function: Observations from 3 species