Vitamin d rescues impaired Mycobacterium tuberculosis-mediated tnf release in hiv+
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- Running Title: Vit D rescues TNF response to MTb in HIV+macrophages
- Infect. Immun. doi:10.1128/IAI.00666-12 IAI Accepts, published online ahead of print on 15 October 2012
- Introduction
- Materials and Methods
- Results 163 Exogenous vitamin D rescues MTb-mediated TNF release in HIV+ human macrophages
- Vitamin D promotes TNF mRNA transcripts in HIV+ human macrophages
- Vitamin D enhancement of TNF release in HIV+ human macrophages is dependent on
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1 macrophages through enhanced TLR signaling pathway in vitro 2 Asha Anandaiah *# , Sanjeev Sinha † , Medhavi Bole *, Surendra K. Sharma † , Narendra Kumar † , 3 Kalpana Luthra † , Xin Li * , Xiuqin Zhou * , Benjamin Nelson * , Xinbing Han, * Souvenir 4 D.Tachado * , Naimish R. Patel * , and Henry Koziel * .
5 * Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Beth Israel 6 Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, MA 7 02215 and † All India Institute of Medical Sciences, 17 SRB wing, Department of Medicine, 3rd 8 floor, New Delhi, India-110029 9
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
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Infect. Immun. doi:10.1128/IAI.00666-12 IAI Accepts, published online ahead of print on 15 October 2012 on September 30, 2017 by guest http://iai.asm.org/ Downloaded from
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17 Mycobacterium tuberculosis (MTb) disease represents an enormous global health problem, with 18
exceptionally high morbidity and mortality in HIV+ persons. Alveolar macrophages from HIV+ 19
persons demonstrate specific and targeted impairment of critical host cell responses including 20
impaired MTb-mediated TNF release and macrophage apoptosis. Vitamin D may promote anti- 21
MTb responses through upregulation of macrophage NO, NADPH oxidase, cathelicidin and 22
autophagy mechanisms, but whether vitamin D promotes anti-MTb mechanisms in HIV+ 23
macrophages is not known. In the current study, human macrophages exposed to MTb 24
demonstrated robust release of TNF, IκB degradation and NF-κB nuclear translocation, and these 25
responses were independent of vitamin D pretreatment. In marked contrast, HIV+U1 human 26
macrophages exposed to MTb demonstrated very low TNF release, and no significant IκB 27
degradation or NF-κB nuclear translocation, whereas vitamin D pretreatment restored these 28
critical responses. The vitamin D-mediated restored responses were in part dependent on 29
macrophage CD14 expression. Importantly, similar response patterns were observed with 30
clinically relevant human alveolar macrophages from healthy individuals and asymptomatic 31
HIV+ persons at high clinical risk of MTb infection. Taken together with the observation that 32
local BALF levels of vitamin D are severely deficient in HIV+ persons, this study demonstrates 33
that exogenous vitamin D can selectively rescue impaired critical innate immune responses in 34
vitro in alveolar macrophages from HIV+ persons at risk for MTb disease, and supports a 35
potential role for exogenous vitamin D as a therapeutic adjuvant in MTb infection in HIV+ 36
persons. 37
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39 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 41
with exceptional morbidity and mortality, especially with MDR or XDR tuberculosis (47)(1). 42
However, the underlying predisposing mechanisms, particularly in HIV+ persons with relatively 43
preserved CD4+ T-lymphocyte counts remain incompletely understood (26, 40, 49). Alveolar 44
macrophages represent a critical cell in the host defense response to MTb (13), and alveolar 45
macrophages from HIV+ persons demonstrate specific and targeted impairment of critical host 46
cell responses including impaired MTb-mediated TNF release and macrophage apoptosis (31) 47
which may be in part related to IL-10 mediated upregulation of BCL3 (30) . Preliminary data 48
suggest that MTb-mediated macrophage apoptosis may be restored by exogenous TNF, 49
suggesting alveolar macrophages from HIV+ persons are not irreversibly impaired and may be 50
responsive to immunomodulation (31). 51
Vitamin D deficiency is associated with susceptibility to MTb disease (5, 6, 48)(22), although 52
the basic underlying mechanisms remain poorly understood. Early in vitro observations 53
demonstrated that exogenous vitamin D suppressed MTb growth in macrophages (4, 36). 54
Vitamin D may promote anti-MTb responses through upregulation of NO (35), NADPH oxidase 55
(38, 39), cathelicidin (20, 21, 50), and autophagy (50) mechanisms in murine models and human 56
macrophages. However, the effect of vitamin D on critical human alveolar macrophage host 57
defense responses has not been fully investigated, and the influence of vitamin D on HIV+ 58
macrophages is not known. 59
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60 defense responses in vitro, focusing on Toll-like receptor (TLR) signaling pathways, as TLRs 61 represent critical innate immune host defense molecules in the recognition of pathogens, 62 including MTb(12, 24, 43) . Furthermore, recognizing the frequent finding of vitamin D 63 deficiency among HIV+ persons (27, 44) (10), this study also focuses on HIV+ macrophages to 64 determine whether exogenous vitamin D can rescue impaired host defense responses to MTb, 65 using human macrophage cell lines and clinically relevant alveolar macrophages. This study 66 demonstrates that exogenous vitamin D can rescue impaired MTb-mediated TNF release in 67 HIV+ macrophages through enhanced TLR and restored IκB/NF-κB signaling; the mechanism of 68 vitamin D mediated rescue of restored responses was in part dependent on macrophage CD14. 69 on September 30, 2017 by guest http://iai.asm.org/ Downloaded from
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70 Human macrophages: (A) Human macrophage cell lines. As a model for study of the influence 71 of HIV infection on human macrophage function, experiments used human monocyte U937 72 (American Tissue Cell Company, ATCC) and HIV-infected human monocyte U1 (subclone of 73 U937; AIDS Research and Reference Reagent Program, Bethesda, MD) cell lines as previously 74 published (31, 41, 42). U1 cells contain two integrated copies of HIV-1 proviral DNA, and are 75 characterized by low levels of constitutive viral expression (7)that can be modulated with 76 specific cytokines and PMA (8). Human U937 and U1 cells were cultured in complete RPMI 77 1640 medium (10% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml 78 streptomycin), except for experiments using live mycobacteria where ceftriaxone (1 µg/ml) was 79 substituted for streptomycin. Cells were harvested during exponential growth phase, washed, and 80 then differentiated into macrophages using PMA (100 nM) at 37°C in 5% CO 2 for 24 h, washed 81 three times with PBS, and incubated an additional 24 h before use. 82 (B) Human alveolar macrophages. For select experiments, human alveolar macrophages were 83 used to confirm critical results observed in cell lines. Prospectively recruited healthy and 84 asymptomatic HIV-seropositive (HIV + ) volunteers had no evidence of active pulmonary disease 85 and had normal spirometry. Healthy individuals had no known risk factors for HIV infection and 86 were confirmed to be HIV seronegative by ELISA, which was performed according to the 87 instructions of the manufacturer (Abbott Diagnostics). Asymptomatic HIV + subjects had a CD4 88 T cell count of >200 cells/mm 3 , undetectable serum viral load (<50 HIV-1 RNA copies/ml), 89 were on HAART or no therapy, and had no history of opportunistic pneumonia. Lung immune 90 cells were obtained by BAL using standard technique (15). All procedures were performed on 91 adult volunteers after informed consent following protocols approved by the Beth Israel 92 on September 30, 2017 by guest http://iai.asm.org/ Downloaded from
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93 BAL fluid and AMs were isolated by adherence for ≥72 h to plastic-bottom tissue culture plates 94 as previously described(15). Isolation of AM from all healthy and HIV + persons yielded cells 95 which were ≥98% viable as determined by trypan blue dye exclusion and demonstrated >95% 96 positive nonspecific esterase staining (15). 97 Microbial organisms and reagents: Virulent (H37Rv) M. tuberculosis, irradiated, was a generous 98 gift from J. Belisle (Colorado State University, Fort Collins, CO) and the National Institute of 99 Allergy and Infectious Diseases Tuberculosis Research materials contract N01-AI-75320). M. 100 bovis (BCG Pasteur) was obtained from ATCC. Stocks were thawed, vortexed, and sonicated 101
using a bath sonicator for 15 s at 500 W and allowed to stand for 10 min, and the upper 200 µl of 102
solution were used for experiments (37). Lipid A (TLR4 ligand) from E. coli F583 Rd mutant 103
and phorbol myristic acid (PMA) were purchased from Sigma Chemical Company (St Louis, 104
MO). Pam 3 Cys-Ser-(Lys) 4 Hydrochloride (PamCys) (TLR3 ligand) was purchased from 105 Calbiochem (San Diego, CA), and 19kDa lipoprotein from M. tuberculosis (TLR2/1 ligand) was 106 purchased from EMC Microcollections (Tuebingen, Germany). 1-pyrrolidinecarbodithioic acid 107 (PDTC), an inhibitor of NF-κB activation, was purchased from Calbiochem (San Diego, CA). 108 1,25(OH)
2 Vitamin D 3 was purchased from Calbiochem (San Diego, CA), and used at a 109 concentration of 100nM unless otherwise specified. 110 RNA isolation and RT-PCR: Total RNA was isolated from macrophages using RNEasy Kit 111 (Qiagen, Valencia, CA) and RT-PCR was performed according to the manufacturer’s protocol 112 Thermoscript PCR system (Invitrogen Life Technologies). The following primers were used: For 113 Vitamin D Receptor (VDR): 5-GCC CAC CAT AAG ACC TAC GA-3 and 5-AGA TTG GAG 114 AAG CTG GAC GA-3. 115 on September 30, 2017 by guest http://iai.asm.org/ Downloaded from
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116 TGT GCT CT-3 and GGA AAC GGT GGC ACA GGA C-3 with Taqman probe 5-TTC CTG 117 CTG ATC CTG CTC ACG GG-3. For TLR4: 5-TGT TGT GGT GTC CCA GCA CT-3 and 5- 118 CTG CCA GGT CTG AGC AAT CTC-3 with Taqman probe 5-CAT CCA GAG CCG CTG 119 GTG TAT CTT TGA A-3. For TNFα: 5'-GGT GCT TGT TCC TCA GCC TC-3' and 5'-CAG 120 GCA GAA GAG CGT GGT G-3' with Taqman probe 5- CTC CTT CCT GAT CGT GGC AGG 121 CG-3. For VDR: 5’-AAG GAC AAC CGA CGC CAC T-3’ and 5’-ATC ATG CCG ATG TCC 122 ACA CA-3’ with Taqman probe 5’-CAG GCC TGC CGG CTC AAA CG-3’. 123
Cytokine detection in cultured supernatants by ELISA: Isolated adherent macrophages (24-well 125
plate, 5 x 10 5 cells/well) are incubated with MTb or BCG (MOI 10:1), for 24 h in the presence or 126 absence of 1,25(OH) 2 Vitamin D (10 -7 M, added for 24 hours prior to MTb or BCG) at 37°C in 127 humidified 5% CO 2 . For select experiments, neutralizing anti-CD14 antibody or IgG1 isotype 128 control (R&D Systems) was added 30 minutes prior to MTb. Culture supernatants were 129 harvested and centrifuged to remove cellular debris, and aliquots were assayed immediately or 130 stored at –80°C until assayed. Specific immunoreactivity to TNF-α (R&D Systems) was 131 measured by ELISA, as described previously (42) 132 Flow cytometry surface receptor analysis: TLR2, TLR4, and CD14 expression was measured via 133 surface antibody labeling (TLR2-PE, TLR4-PE from Invivogen; CD14-PE from MACS) in 134 macrophage cell suspensions with a Cytomics FC500 flow cytometer (Beckman Coulter) as 135 previously published (42). Results were recorded as mean relative fluorescence units (RFU) and 136 the percentage of the population staining positive. 137 on September 30, 2017 by guest http://iai.asm.org/ Downloaded from
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138 buffer with protease and phosphatase inhibitors. Western blotting was performed utilizing a 139 standard protocol (51) with antibodies specific to IKBα and β–actin (Cell Signaling Technology). 140 Resolved bands were quantified by densitometry (Ambersham Biosciences), and results 141 expressed as relative units (RU). 142 NF-κB ELISA: Adherent isolated macrophages (6 well plate, 3 x 10 6 cells/well) were incubated 143 with MTb for 0–120 min, macrophage nuclear extracts were prepared by using the NE-PER kit 144 (Pierce) according to the manufacturer’s protocol, and ELISA specific for p65 was performed 145 using the Transfactor NF-κB p65 Colorimetric kit according to manufacturer’s protocol 146 (Clontech). Protein loading was standardized using Bradford assay (Bio-Rad). 147 Serum and BAL Vitamin D measurements: Archived frozen clinical samples of paired 148 bronchoalveolar lavage fluid (BALF) and serum (stored at -80 o C) were available from four 149 groups of patients who underwent bronchoscopy at the All India Institute of Medical Sciences, 150 (New Delhi, India): 1) HIV seronegative without MTb; 2) HIV seronegative with 151 microbiologically confirmed active MTb; 3) HIV+ without MTb; and 4) HIV+ with 152 microbiologically confirmed MTb disease. Patients provided informed consent and the study 153 protocol was approved by the AIIMS Ethics Committee. 25(OH)Vitamin D 3 and
154 1,25(OH)
2 Vitamin D 3 levels were measured in paired serum and BALF samples by ELISA 155 according to manufacturer's protocol (IDS Ltd., Fountain Hills,
AZ). Vitamin D levels were 156 normalized for BAL-associated dilution factor using urea nitrogen measurements as previously 157 described (16),(34). 158 on September 30, 2017 by guest http://iai.asm.org/ Downloaded from
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159 test), and a p < 0.05 was considered to be significant. Experiments were repeated a minimum of 160 three times. 161 162
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163 Exogenous vitamin D rescues MTb-mediated TNF release in HIV+ human macrophages 164
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 166
robust increase in TNF release in response to MTb (Fig 1a), and 1,25(OH) 2 vitamin D 3 (1,25D
3 ) 167 pretreatment did not influence macrophage TNF release constitutively or in response to MTb 168
challenge (Fig 1a). In HIV+U1 macrophages, constitutive TNF release was also low, but TNF 169
release in response to MTb was significantly impaired compared to U937 cells (Fig 1b), 170
consistent with prior publications (31). However, in marked contrast to U937 cells, pretreatment 171
of HIV+U1 macrophages with 1,25D 3 dramatically increased macrophage TNF release in 172 response to MTb in a concentration-dependent manner, to levels comparable to U937 173 macrophages (Fig1b,c), whereas 1,25D 3 pretreatment did not influence constitutive TNF release 174 in HIV+U1 macrophages. Thus, exogenous 1,25D 3 selectively restored impaired MTb-mediated 175 TNF release in HIV+ human macrophages. 176
177 Vitamin D promotes TNF mRNA transcripts in HIV+ human macrophages 178
The main biological actions of vitamin D occur following conversion of the principle circulating 179
25(OH)D 3 (25D 3 )
form to 1,25D 3 by the cellular enzyme 1-alpha hydroxylase CYP27B1, and 180 subsequent binding to the intracellular vitamin D receptor (VDR) (46). Although the main site 181 of CYP27B1 hydroxylase expression is the kidney, immune cells including macrophages express 182 CYP27B1 hydroxylase and thus are able to independently convert 25D 3 to biologically active 183 1,25D
3 (25). In the current study both human U937 and HIV+U1 macrophages expressed 184 on September 30, 2017 by guest http://iai.asm.org/ Downloaded from
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185 differences in 1,25D 3 -mediated macrophage responses were not attributable to significant 186 differences in levels of VDR. To determine the mechanism for 1,25D 3 rescue of TNF release in 187 HIV+ macrophages, we next examined TNF mRNA levels. Exogenous 1,25D 3 pretreatment did 188 not influence TNF mRNA levels in human U937 macrophages (Fig 2b) whereas TNF mRNA 189 levels were significantly increased by 1,25D 3 in human HIV+U1 macrophages in response to 190 mycobacteria (Fig 2c). These results suggest that increased MTb-mediated TNF release in 191 HIV+U1 macrophages was associated with increased TNF mRNA. 192
Vitamin D enhancement of TNF release in HIV+ human macrophages is dependent on 194
recognition of known TLR ligands 195
Toll-like receptor 2 (TLR2) and TLR4 are critical host defense signaling molecules that mediate 196
TNF-release by macrophages in response to MTb (33). In the current study, human U937 197
macrophages released TNF in response to TLR2 and TLR4 agonists, with significant change 198
following 1,25D 3 pretreatment only to Lipid A (Fig 3a), consistent with prior observations (32). 199 In contrast, 1,25D 3 pretreatment of human HIV+U1 macrophages significantly increased TNF 200 release in response to multiple TLR2 and TLR4 ligands (Fig 3b), including the MTb 19 kDa 201 lipopeptide (recognized by TLR2/1). Both TLR2 and TLR4 mRNA and surface expression were 202 comparable in human U937 and HIV+U1 macrophages, and TLR2 and TLR4 levels were not 203 significantly altered by 1,25D 3 pretreatment (Fig 3c,d). Thus, the observed rescue of MTb- 204 mediated TNF release following 1,25D 3 pretreatment was not associated with significant 205 alteration in mRNA or surface expression of macrophage TLR2 or TLR4 molecules. 206 on September 30, 2017 by guest http://iai.asm.org/ Downloaded from
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