Vitamin d rescues impaired Mycobacterium tuberculosis-mediated tnf release in hiv+


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Vitamin D rescues impaired Mycobacterium tuberculosis-mediated TNF release in HIV+ 





macrophages through enhanced TLR signaling pathway in vitro 

Asha Anandaiah



*#

, Sanjeev Sinha

, Medhavi Bole



*, 

Surendra K. Sharma

, Narendra Kumar





Kalpana Luthra

, Xin Li



*

, Xiuqin Zhou

*

, Benjamin Nelson



*

, Xinbing Han,

*

 Souvenir 



D.Tachado

*

, Naimish R. Patel



 *

, and Henry Koziel

.  


*

Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Beth Israel 



Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, MA 

02215 and 



 All India Institute of Medical Sciences, 17 SRB wing, Department of Medicine, 3rd 

floor,  New Delhi, India-110029 



 

10 



Running Title: Vit D rescues TNF response to MTb in HIV+macrophages 

11 


 

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Corresponding Author: Asha Anandaiah 

13 


Email: aanandai@bidmc.harvard.edu 

14 


 

15 


16 

 

Copyright © 2012, American Society for Microbiology. All Rights Reserved.



Infect. Immun. doi:10.1128/IAI.00666-12 

IAI Accepts, published online ahead of print on 15 October 2012

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 Abstract 



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Mycobacterium tuberculosis (MTb) disease represents an enormous global health problem, with 

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exceptionally high morbidity and mortality in HIV+ persons.  Alveolar macrophages from HIV+ 

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persons demonstrate specific and targeted impairment of critical host cell responses including 

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impaired MTb-mediated TNF release and macrophage apoptosis.  Vitamin D may promote anti-

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MTb responses through upregulation of macrophage NO, NADPH oxidase, cathelicidin and 

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autophagy mechanisms, but whether vitamin D promotes anti-MTb mechanisms in HIV+ 

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macrophages is not known.  In the current study, human macrophages exposed to MTb 

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demonstrated robust release of TNF, IκB degradation and NF-κB nuclear translocation, and these 

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responses were independent of vitamin D pretreatment.  In marked contrast, HIV+U1 human 

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macrophages exposed to MTb demonstrated very low TNF release, and no significant IκB 

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degradation or NF-κB nuclear translocation, whereas vitamin D pretreatment restored these 

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critical responses.  The vitamin D-mediated restored responses were in part dependent on 

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macrophage CD14 expression.  Importantly, similar response patterns were observed with 

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clinically relevant human alveolar macrophages from healthy individuals and asymptomatic 

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HIV+ persons at high clinical risk of MTb infection.  Taken together with the observation that 

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local BALF levels of vitamin D are severely deficient in HIV+ persons, this study demonstrates 

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that exogenous vitamin D can selectively rescue impaired critical innate immune responses in 

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vitro in alveolar macrophages from HIV+ persons at risk for MTb disease, and supports a 

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potential role for exogenous vitamin D as a therapeutic adjuvant in MTb infection in HIV+ 

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persons. 

37 


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Introduction 



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Mycobacterium tuberculosis (MTb) infection in HIV+ persons represents an enormous global 

40 


health problem, frequently occurs in persons in early stages of HIV disease, and is associated 

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with exceptional morbidity and mortality, especially with MDR or XDR tuberculosis (47)(1).  

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However, the underlying predisposing mechanisms, particularly in HIV+ persons with relatively 

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preserved CD4+ T-lymphocyte counts remain incompletely understood (26, 40, 49).  Alveolar 

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macrophages represent a critical cell in the host defense response to MTb (13), and alveolar 

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macrophages from HIV+ persons demonstrate specific and targeted impairment of critical host 

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cell responses including impaired MTb-mediated TNF release and macrophage apoptosis (31) 

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which may be in part related to IL-10 mediated upregulation of BCL3 (30) .  Preliminary data 

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suggest that MTb-mediated macrophage apoptosis may be restored by exogenous TNF, 

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suggesting alveolar macrophages from HIV+ persons are not irreversibly impaired and may be 

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responsive to immunomodulation (31). 

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Vitamin D deficiency is associated with susceptibility to MTb disease (5, 6, 48)(22), although 

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the basic underlying mechanisms remain poorly understood. Early in vitro observations 

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demonstrated that exogenous vitamin D suppressed MTb growth in macrophages (4, 36).  

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Vitamin D may promote anti-MTb responses through upregulation of NO (35), NADPH oxidase 

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(38, 39), cathelicidin (20, 21, 50), and autophagy (50) mechanisms in murine models and human 

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macrophages.   However, the effect of vitamin D on critical human alveolar macrophage host 

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defense responses has not been fully investigated, and the influence of vitamin D on HIV+ 

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macrophages is not known. 

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The purpose of this study is to examine the influence of vitamin D on human macrophage host 



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defense responses in vitro, focusing on Toll-like receptor (TLR) signaling pathways, as TLRs 

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represent critical innate immune host defense molecules in the recognition of pathogens, 



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including MTb(12, 24, 43) .  Furthermore, recognizing the frequent finding of vitamin D 

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deficiency among HIV+ persons (27, 44) (10), this study also focuses on HIV+ macrophages to 



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determine whether exogenous vitamin D can rescue impaired host defense responses to MTb

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using human macrophage cell lines and clinically relevant alveolar macrophages.  This study 



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demonstrates that exogenous vitamin D can rescue impaired MTb-mediated TNF release in 

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HIV+ macrophages through enhanced TLR and restored IκB/NF-κB signaling; the mechanism of 



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vitamin D mediated rescue of restored responses was in part dependent on macrophage CD14.

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Materials and Methods: 



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Human macrophages:  (A) Human macrophage cell lines.  As a model for study of the influence 

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of HIV infection on human macrophage function, experiments used human monocyte U937 



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(American Tissue Cell Company, ATCC) and HIV-infected human monocyte U1 (subclone of 

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U937; AIDS Research and Reference Reagent Program, Bethesda, MD) cell lines as previously 



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published (31, 41, 42). U1 cells contain two integrated copies of HIV-1 proviral DNA, and are 

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characterized by low levels of constitutive viral expression (7)that can be modulated with 



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specific cytokines and PMA (8).  Human U937 and U1 cells were cultured in complete RPMI 

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1640 medium (10% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml 



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streptomycin), except for experiments using live mycobacteria where ceftriaxone (1 µg/ml) was 

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substituted for streptomycin. Cells were harvested during exponential growth phase, washed, and 



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then differentiated into macrophages using PMA (100 nM) at 37°C in 5% CO

2

 for 24 h, washed 



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three times with PBS, and incubated an additional 24 h before use.  

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(B) Human alveolar macrophages. For select experiments, human alveolar macrophages were 



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used to confirm critical results observed in cell lines.  Prospectively recruited healthy and 

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asymptomatic HIV-seropositive (HIV



+

) volunteers had no evidence of active pulmonary disease 

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and had normal spirometry. Healthy individuals had no known risk factors for HIV infection and 



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were confirmed to be HIV seronegative by ELISA, which was performed according to the 

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instructions of the manufacturer (Abbott Diagnostics). Asymptomatic HIV



+

 subjects had a CD4 

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T cell count of >200 cells/mm



3

, undetectable serum viral load  (<50 HIV-1 RNA copies/ml), 

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were on HAART or no therapy, and had no history of opportunistic pneumonia.  Lung immune 



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cells were obtained by BAL using standard technique (15).  All procedures were performed on 

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adult volunteers after informed consent following protocols approved by the Beth Israel 



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Deaconess Medical Center Institutional Review Board. The cells were separated from the pooled 



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BAL fluid and AMs were isolated by adherence for ≥72 h to plastic-bottom tissue culture plates 

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as previously described(15). Isolation of AM from all healthy and HIV



+

 persons yielded cells 

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which were ≥98% viable as determined by trypan blue dye exclusion and demonstrated >95% 



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positive nonspecific esterase staining (15).  

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Microbial organisms and reagents: Virulent (H37Rv) M. tuberculosis, irradiated,  was a generous 



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gift from J. Belisle (Colorado State University, Fort Collins, CO) and the National Institute of 

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Allergy and Infectious Diseases Tuberculosis Research materials contract N01-AI-75320). M. 



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bovis (BCG Pasteur) was obtained from ATCC.  Stocks were thawed, vortexed, and sonicated 

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using a bath sonicator for 15 s at 500 W and allowed to stand for 10 min, and the upper 200 µl of 

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solution were used for experiments (37).  Lipid A (TLR4 ligand) from E. coli F583 Rd mutant 

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and phorbol myristic acid (PMA) were purchased from Sigma Chemical Company (St Louis, 

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MO). Pam

3

Cys-Ser-(Lys)



Hydrochloride (PamCys) (TLR3 ligand) was purchased from 

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Calbiochem (San Diego, CA), and 19kDa lipoprotein from M. tuberculosis (TLR2/1 ligand) was 



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purchased from EMC Microcollections (Tuebingen, Germany). 1-pyrrolidinecarbodithioic acid 

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(PDTC), an inhibitor of NF-κB activation, was purchased from Calbiochem (San Diego, CA). 



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1,25(OH)


2

Vitamin D

3

 was purchased from Calbiochem (San Diego, CA), and used at a 



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concentration of 100nM unless otherwise specified. 

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RNA isolation and RT-PCR: Total RNA was isolated from macrophages using RNEasy Kit 



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(Qiagen, Valencia, CA) and RT-PCR was performed according to the manufacturer’s protocol 

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Thermoscript PCR system (Invitrogen Life Technologies). The following primers were used: For 



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Vitamin D Receptor (VDR): 5-GCC CAC CAT AAG ACC TAC GA-3 and 5-AGA TTG GAG 

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AAG CTG GAC GA-3.   



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Real-time PCR: was performed using the following primers. For TLR2: 5-TCT GGC ATG TGC 



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TGT GCT CT-3 and GGA AAC GGT GGC ACA GGA C-3 with Taqman probe 5-TTC CTG 

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CTG ATC CTG CTC ACG GG-3. For TLR4: 5-TGT TGT GGT GTC CCA GCA CT-3 and 5-



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CTG CCA GGT CTG AGC AAT CTC-3 with Taqman probe 5-CAT CCA GAG CCG CTG 

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GTG TAT CTT TGA A-3. For TNFα: 5'-GGT GCT TGT TCC TCA GCC TC-3' and 5'-CAG 



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GCA GAA GAG CGT GGT G-3' with Taqman probe 5- CTC CTT CCT GAT CGT GGC AGG 

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CG-3. For VDR: 5’-AAG GAC AAC CGA CGC CAC T-3’ and 5’-ATC ATG CCG ATG TCC 



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ACA CA-3’ with Taqman probe 5’-CAG GCC TGC CGG CTC AAA CG-3’. 

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Cytokine detection in cultured supernatants by ELISA: Isolated adherent macrophages (24-well 

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plate, 5 x 10

5

 cells/well) are incubated with MTb or BCG (MOI 10:1), for 24 h in the presence or 



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absence of 1,25(OH)

2

Vitamin D (10



-7

M, added for 24 hours prior to MTb or BCG) at 37°C in 

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humidified 5% CO



2

.  For select experiments, neutralizing anti-CD14 antibody or IgG1 isotype 

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control (R&D Systems)  was added 30 minutes prior to MTb.  Culture supernatants were 



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harvested and centrifuged to remove cellular debris, and aliquots were assayed immediately or 

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stored at –80°C until assayed.  Specific immunoreactivity to TNF-α (R&D Systems) was 



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measured by ELISA, as described previously (42) 

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Flow cytometry surface receptor analysis: TLR2, TLR4, and CD14 expression was measured via 



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surface antibody labeling (TLR2-PE, TLR4-PE from Invivogen; CD14-PE from MACS) in 

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macrophage cell suspensions with a Cytomics FC500 flow cytometer (Beckman Coulter) as 



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previously published (42). Results were recorded as mean relative fluorescence units (RFU) and 

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the percentage of the population staining positive.  



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Western blotting: Cell cytoplasmic protein extracts were prepared using standard ice-cold RIPA 



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buffer with protease and phosphatase inhibitors. Western blotting was performed utilizing a 

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standard protocol (51) with antibodies specific to IKBα and β–actin (Cell Signaling Technology). 



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Resolved bands were quantified by densitometry (Ambersham Biosciences), and results 

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expressed as relative units (RU). 



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NF-κB ELISA:  Adherent isolated macrophages (6 well plate, 3 x 10

6

 cells/well) were incubated 



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with MTb for 0–120 min, macrophage nuclear extracts were prepared by using the NE-PER kit 

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(Pierce) according to the manufacturer’s protocol, and ELISA specific for p65 was performed 



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using the Transfactor NF-κB p65 Colorimetric kit according to manufacturer’s protocol 

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(Clontech). Protein loading was standardized using Bradford assay (Bio-Rad).  



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Serum and BAL Vitamin D measurements: Archived frozen clinical samples of paired 

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bronchoalveolar lavage fluid (BALF) and serum (stored at -80



o

C) were available from four 

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groups of patients who underwent bronchoscopy at the All India Institute of Medical Sciences



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(New Delhi, India):  1) HIV seronegative without MTb; 2) HIV seronegative with 

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microbiologically confirmed active MTb; 3) HIV+ without MTb; and 4) HIV+ with 



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microbiologically confirmed MTb disease.  Patients provided informed consent and the study 

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protocol was approved by the AIIMS Ethics Committee.  25(OH)Vitamin D



3

 and 


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1,25(OH)


2

Vitamin D

3

 levels were measured in paired serum



 

and BALF samples by ELISA 

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according to manufacturer's



 

protocol (IDS Ltd., Fountain Hills,

 

AZ).  Vitamin D levels were 



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normalized for BAL-associated dilution factor using urea nitrogen measurements as previously 

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described (16),(34). 



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Statistical methods  All data was analyzed using nonparametric methodology (Mann-Whitney U 



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test), and a p < 0.05 was considered to be significant.  Experiments were repeated a minimum of 

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three times. 



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Results 



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Exogenous vitamin D rescues MTb-mediated TNF release in HIV+ human macrophages 

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TNF release represents a critical macrophage response to MTb challenge (9).  In the current 

165 


study, unstimulated human U937 macrophages demonstrated low constitutive TNF release and 

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robust increase in TNF release in response to MTb (Fig 1a), and 1,25(OH)

2

 vitamin D



3

 (1,25D


3

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pretreatment did not influence macrophage TNF release constitutively or in response to MTb 

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challenge (Fig 1a).  In HIV+U1 macrophages, constitutive TNF release was also low, but TNF 

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release in response to MTb was significantly impaired compared to U937 cells (Fig 1b), 

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consistent with prior publications (31).  However, in marked contrast to U937 cells, pretreatment 

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of HIV+U1 macrophages with 1,25D

3

 dramatically increased macrophage TNF release in 



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response to MTb in a concentration-dependent manner, to levels comparable to U937 

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macrophages (Fig1b,c), whereas 1,25D



3

 pretreatment did not influence constitutive TNF release 

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in HIV+U1 macrophages.   Thus, exogenous 1,25D



selectively restored impaired MTb-mediated 

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TNF release in HIV+ human macrophages. 



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Vitamin D promotes TNF mRNA transcripts in HIV+ human macrophages 

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The main biological actions of vitamin D occur following conversion of the principle circulating 

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25(OH)D

(25D



3

)

   



form to 1,25D

by the cellular enzyme 1-alpha hydroxylase CYP27B1, and 



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subsequent binding to the intracellular vitamin D receptor (VDR) (46).  Although the main site 

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of CYP27B1 hydroxylase expression is the kidney, immune cells including macrophages express 



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CYP27B1 hydroxylase and thus are able to independently convert 25D

to biologically active 



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1,25D


(25).  In the current study both human U937 and HIV+U1 macrophages expressed 

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mRNA for vitamin D receptor (VDR) at comparable levels (Fig 2a), suggesting observed 



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differences in 1,25D

-mediated macrophage responses were not attributable to significant 



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differences in levels of VDR. To determine the mechanism for 1,25D

 rescue of TNF release in 



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HIV+ macrophages, we next examined TNF mRNA levels.  Exogenous 1,25D

 pretreatment did 



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not influence TNF mRNA levels  in human U937 macrophages (Fig 2b) whereas TNF mRNA 

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levels were significantly increased by 1,25D



 in human HIV+U1 macrophages in response to 

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mycobacteria (Fig 2c).  These results suggest that increased MTb-mediated TNF release in 



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HIV+U1 macrophages was associated with increased TNF mRNA. 

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Vitamin D enhancement  of TNF release in HIV+ human macrophages is dependent on 

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recognition of known TLR ligands 

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Toll-like receptor 2 (TLR2) and TLR4 are critical host defense signaling molecules that mediate 

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TNF-release by macrophages in response to MTb (33).  In the current study, human U937 

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macrophages released TNF in response to TLR2 and TLR4 agonists, with significant change 

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following 1,25D

 pretreatment only to Lipid A (Fig 3a), consistent with prior observations (32).  



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In contrast, 1,25D

 pretreatment of human HIV+U1 macrophages significantly increased TNF 



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release in response to multiple TLR2 and TLR4 ligands (Fig 3b), including the MTb 19 kDa 

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lipopeptide (recognized by TLR2/1).  Both TLR2 and TLR4 mRNA and surface expression were 



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comparable in human U937 and HIV+U1 macrophages, and TLR2 and TLR4 levels were not 

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significantly altered by 1,25D



 pretreatment (Fig 3c,d).  Thus, the observed rescue of MTb-

204 

mediated TNF release following 1,25D



pretreatment was not associated with significant 

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alteration in mRNA or surface expression of macrophage TLR2 or TLR4 molecules. 



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