Vitamin d rescues impaired Mycobacterium tuberculosis-mediated tnf release in hiv+
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- Vitamin D upregulates macrophage CD14 expression
- Reduced BALF vitamin D levels in HIV+ patients with active tuberculosis.
- Acknowledgements
- References
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Upregulation of NF-κB signaling by vitamin D in HIV+ human macrophages 208
The observation that exogenous 1,25D 3 rescue of MTb-mediated TNF release in HIV+ human 209 macrophages was associated with increased TNF mRNA levels, but not associated with 210 alteration of TLR surface expression (major MTb recognition signaling receptors) suggested that 211 signaling pathways downstream of TLR may be modulated by 1,25D 3 . TLR signaling promotes 212 IκB degradation and allows NF-κB nuclear translocation and subsequent host defense gene 213 activation including TNF (2, 28). In the current study, human U937 macrophages demonstrated 214 rapid MTb-mediated IκB degradation, with no significant change with 1,25D 3 pretreatment, but 215 with expected inhibition by PDTC (an inhibitor of IκB degradation) (Fig 4a). In marked 216 contrast, human HIV+U1 macrophages failed to demonstrate significant IκB degradation in 217 response to MTb over time (Fig 4a), but exogenous 1,25D 3 pretreatment promoted MTb- 218 mediated IkB degradation, although less robust and with delayed kinetics compared to U937 219 cells (Fig 4a). Consistent with these findings, using an independent assay human U937 220 macrophages demonstrated NF-κB nuclear translocation in response to MTb, but no further 221 increase in NF-κB nuclear translocation with 1,25D 3 pretreatment (Fig 4b). In marked contrast, 222 human HIV+U1 macrophages demonstrated limited NF-κB nuclear translocation in response to 223 MTb, but dramatic increase in MTb-mediated NF-κB nuclear translocation upon pretreatment 224 with 1,25D 3 (Fig 4b). Thus, 1,25D 3 selectively promoted MTb-mediated IκB degradation and 225 NF-κB nuclear translocation in human HIV+U1 macrophages. 226
Vitamin D upregulates macrophage CD14 expression 228
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229 macrophages) that facilitates TLR ligand binding (29). 1,25D 3 upregulates CD14 expression in 230 human monocytes (19), and could enhance TLR signaling. In the current study, constitutive 231 CD14 surface expression was relatively low for both human U937 and HIV+U1 human 232 macrophages, and 1,25D 3 pretreatment significantly increased CD14 surface expression in both 233 populations of macrophages (Fig 5a). In human U937 macrophages pretreated with 1,25D 3 ,
MTb-mediated TNF release was not significantly altered in the presence of anti-CD14 235
neutralizing antibody (Fig 5b), whereas LPS-mediated TNF release was markedly reduced by 236
anti-CD14 antibody, as expected. However, in marked contrast, in HIV+U1 macrophages 237
pretreated with 1,25D 3 , MTb-mediated TNF release was significantly reduced in the presence of 238 anti-CD14 neutralizing antibody (Fig 5c). Thus, although 1,25D 3 upregulated macrophage 239 CD14 surface expression in both U937 and HIV+U1 cells, CD14 upregulation contributed to 240 1,25D
3 -mediated rescue of MTb-mediated TLR signaling in HIV+U1 macrophages, whereas 241 TNF release was CD14 independent in U937 macrophages. 242
243 Vitamin D rescues MTb-mediated TNF release in human alveolar macrophages. 244
To validate the above findings, select experiments were next performed using clinically relevant 245
human alveolar macrophages. Consistent with the results using human macrophage cell lines, 246
human alveolar macrophages from healthy individuals demonstrated significant release of TNF 247
in response to MTb or BCG, but without significant influence following 1,25D 3 pretreatment 248 (Fig 6a), whereas 1,25D 3 pretreatment significantly increased MTb-mediated TNF release in 249 alveolar macrophages from asymptomatic HIV+ persons (Fig 6a), even in immune reconstituted 250 on September 30, 2017 by guest http://iai.asm.org/ Downloaded from
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251 cell lines, TLR2 and TLR4 mRNA (Fig 6b) and surface expression (Fig 6c) were comparable in 252 human alveolar macrophages from healthy and from asymptomatic HIV+ persons and TLR 253 expression was not influenced by 1,25D 3 (Fig 6b,c). Although 1,25D 3 upregulated CD14 254 expression in alveolar macrophages from both healthy and HIV+ persons (Fig 7a), in alveolar 255 macrophages from asymptomatic HIV+ persons pretreated with 1,25D 3 , neutralizing anti-CD14 256 antibody significantly reduced MTb-mediated TNF release (Fig 7b), whereas neutralizing anti- 257 CD14 antibody had no effect on alveolar macrophages from healthy persons (data not shown). 258 Collectively, these experiments validate the results observed with human U937 and HIV+U1 259 macrophages, and suggest that 1,25D 3 may selectively rescue MTb-mediated TNF release in 260 alveolar macrophages from HIV+ persons, in part through a CD14-dependent mechanism. 261
262 Reduced BALF vitamin D levels in HIV+ patients with active tuberculosis. 263
Serum levels of 25D 3 are reduced in persons with active tuberculosis (6, 48), and HIV infection 264 is associated with reduced serum levels of 25D 3 (10, 27, 44). However, vitamin D levels in the 265 lungs of persons with HIV or HIV-MTb coinfection have not been reported. In the current study, 266 biologically active 1,25D 3 was not detected in any cell-free bronchoalveolar lavage fluid 267 (BALF) specimen (data not shown). In contrast, 25D 3 levels were readily detected in the BALF 268 of all persons, but were lowest in persons with HIV infection, and especially in HIV infected 269 persons with active MTb disease (Fig 7c). These data suggest that HIV infection is associated 270 with local vitamin D deficiency in the alveolar airspace, especially in HIV+ persons co-infected 271 with M. tuberculosis. 272 273
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274 This study shows that exogenous 1,25D 3 rescues MTb-mediated TNF release in HIV+ human 275 macrophages. In the absence of HIV infection, human macrophages exposed to MTb 276 demonstrated a robust release of TNF, IκB degradation and NF-κB nuclear translocation, and 277 these responses were independent of 1,25D 3 pretreatment. In marked contrast, HIV+U1 human 278 macrophages exposed to MTb demonstrated very low TNF release, and no significant IκB 279 degradation or NF-κB nuclear translocation, but with significant rescue of these responses with 280 1,25D
3 pretreatment. Furthermore, the 1,25D 3 mediated rescue of macrophage function in 281 response to MTb was dependent in part on CD14 expression. Importantly, similar response 282 patterns were observed with clinically relevant human alveolar macrophages from healthy 283 individuals and asymptomatic HIV+ persons at high clinical risk of MTb infection. Taken 284 together, these data support the concept that 1,25D 3 pretreatment rescues impaired MTb- 285 mediated TNF release in HIV+ macrophages through restored IκB/NF-κB signaling that is in 286 part CD14-dependent. 287
This is the first study, to our knowledge, to examine the immunomodulatory effects of 289
exogenous vitamin D on the response of HIV+ macrophages to MTb. The clinical implications 290
of the current investigation is of particular importance recognizing that the global MTb epidemic 291
disproportionately affects HIV+ persons. Epidemiologic data show that unlike other 292
opportunistic infections, the risk of MTb disease rises soon after HIV seroconversion despite 293
relatively preserved CD4 counts and is not completely reversed by HAART (17, 40). Previous 294
studies from our laboratory and other investigators demonstrate that HIV is associated with 295
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296 including intracellular signaling, chemokine production, TNFα and other proinflammatory 297 cytokine release, and macrophage apoptosis (31, 37), which may in part contribute to the 298 elevated risk of MTb disease in the absence of significantly reduced circulating CD4 T- 299 lymphocte counts. In the current study, macrophage innate immune function was restored by 300 exogenous 1,25D 3 . Specifically, in HIV+ macrophages exogenous 1,25D 3 restored TNF release, 301 upregulated TNF mRNA, enhanced TLR2 and TLR4 responses, and rescued IκB degradation 302 and NF-κB nuclear translocation. Furthermore, these 1,25D 3 -restored host defense responses 303 were dependent on CD14 expression in HIV+ macrophages. Taken together, these findings 304 support the concept that vitamin D may selectively restore TLR signaling, a critical recognition 305 signaling pathway in the host cell response to MTb challenge. 306
The mechanism for vitamin D rescue of macrophage innate function in HIV+ macrophages is 308
through TLR signaling. The differences in influence of 1,25D 3 on U937 and HIV+ U1 309 macrophages was not explained by obvious differences in levels of the principal receptor for 310 vitamin D, VDR, which were similar in the U937 and HIV+U1, and human alveolar 311 macrophages. Furthermore, the findings that TLR2 and TLR4 ligand-mediated TNF release 312 were enhanced by vitamin D (including TLR2 ligand 19kD MTb lipoprotein-mediated TNF 313 release), and that the IkB/NF-kB pathway was restored in HIV+U1 macrophages while 314 constitutive surface expression of TLR2 and TLR4 were similar and without significant 315 alterations in response to 1,25D 3 suggests that 1,25D 3 stimulates other components of the TLR 316 signaling pathway in HIV+ macrophages. Finally, the finding that 1,25D 3 upregulates the TLR 317 co-receptor CD14, and that neutralizing CD14 in HIV+ macrophages pretreated with 1,25D 3
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319 modulation of TLR co-receptor CD14, whereas in the absence of HIV infection, MTb-mediated 320 TNF release is mediated through IκB/NF-κB signaling but is CD14-independent. CD14- 321 independence of MTb-mediated TNF release in healthy cells may be due to activation of 322 alternate pathways or expression of alternate costimulatory molecules that may be suppressed in 323 HIV infected cells, or perhaps other mechanisms. Determining the specific pathways involved in 324 the macrophage response to MTb represents an area of active investigation. 325
326 In the current study, the mechanism of rescued MTb-mediated TNF release in HIV+ 327
macrophages was in part attributed to CD14 expression or signaling. However, other host 328
defense receptors and signaling pathways may also contribute, but were not specifically 329
investigated. Although 1,25D 3 rescued MTb-mediated human HIV+ macrophage TNF release, 330 the influence on other cytokines and other macrophage host defense functions were not 331 investigated. Other limitations of the current study include the experimental design that 332 examined 1,25D 3 pretreatment, but did not examine the influence of 1,25D 3 on macrophages 333 previously (or simultaneously) infected with MTb. Although 25D 3 levels were very low in 334 BALF from HIV+ persons, especially in persons co-infected with MTb, detailed clinical 335 characteristics, specific correlation with serum 1,25D 3 levels, and correlation with macrophage 336 function for individuals were not available. The use of human macrophage cell lines may not 337 reflect behavior of primary human macrophages, although the consistent findings of similar 338 response patterns in human alveolar macrophages in both the current (although the number of 339 subjects was limited) and previous studies (18, 31, 41) validates these observations and supports 340 the use of these human macrophages cell lines as an experimental model. Differences in the 341 on September 30, 2017 by guest http://iai.asm.org/ Downloaded from
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342 alveolar macrophages from HIV+ persons may in part reflect differences in the level of HIV 343 infection (as 100% of U1 macrophages contain HIV genome, whereas <10% of human alveolar 344 macrophages contain HIV genome) (16, 51) (31, 42). The use of irradiated virulent MTb may 345 not accurately predict the influence of live MTb on human macrophage function, although we 346 previously observed similar human macrophage TNF responses comparing irradiated to live 347 H37Rv MTb (30, 31). The use of irradiated MTb did not allow determination of influence of 348 1,25D
3 on MTb growth. Finally, in vitro experiments may not accurately reflect in vivo 349 behavior, although the inclusion of clinically relevant primary human alveolar macrophages may 350 allow more direct translation of these findings to human disease. Our data provide the rationale 351 for further study, including further validation using alveolar macrophages from a larger number 352 of HIV+ persons. 353
Our results are consistent with several earlier studies that showed a stimulatory effect of 1,25D 3
355 on monocyte-macrophage responses to MTb including respiratory burst, autophagy, and 356 antimicrobial protein production (21, 39, 50). Our findings of a select benefit of exogenous 357 1,25D
3 on HIV+ human macrophages (but not healthy macrophages) is consistent with one 358 previous study which showed that 1,25D 3 suppressed replication of Mycobacterium avium 359 (MAC) in macrophages from HIV+ subjects, but had no effect in macrophages from healthy 360 individuals (11). These observations suggest that the innate immune modulatory effects of 361 exogenous 1,25D 3 are further modulated in the setting of HIV infection. HIV does not appear to 362 grossly alter macrophage VDR expression. Other possible explanations for differences in 363 measured responses of human HIV+ macrophage to exogenous 1,25D 3 include differences in 364 on September 30, 2017 by guest http://iai.asm.org/ Downloaded from
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365 receptor expression to critical or threshold levels to activate signaling pathways, or differences in 366 activation states of HV+ macrophages compared to macrophages from healthy persons (3), 367 although these were not specifically investigated in the current study. 368
The potential benefit of vitamin D supplementation in the treatment of MTb disease in HIV+ 370
persons has not yet been established. To date, two clinical trials have investigated the effect of 371
vitamin D supplementation on MTb disease, and neither demonstrated significant benefit. 372
However, neither trial included significant numbers of HIV+ patients. Furthermore, both trials 373
investigated vitamin D as an adjunctive therapy to antimicrobials in the treatment of established 374
active MTb (23, 45). Our central observation is that vitamin D pretreatment can rescue defective 375
MTb-mediated TNF release in HIV+ human macrophages. Clinically, TNF is crucial to 376
maintaining latency in MTb-infected individuals as evidenced by the high incidence of 377
reactivation MTb seen in patients treated with anti-TNF strategies (14). Our observation that 378
vitamin D augments the MTb-mediated TNF response suggests that vitamin D supplementation 379
may be more effective in preventing MTb disease in HIV+ individuals, rather than as a primary 380
treatment for active MTb infection, although this hypothesis has yet to be clinically investigated. 381
382
In conclusion, exogenous Vitamin D rescues MTb-mediated TNF release in HIV+ macrophages 383
by restoring TLR-mediated NF-kB signaling in part through a CD14-dependent mechanism, 384
whereas vitamin D does not influence MTb-mediated TNF release in healthy macrophages. 385
These data further support the important concept that alveolar macrophages from HIV+ persons 386
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387 lymphocyte counts >200 and undetectable viral load) continue to exhibit evidence of intrinsic 388 macrophage dysfunction, suggesting that HAART is not sufficient to restore macrophage innate 389 function. Furthermore, this study supports the concept that macrophages from HIV+ persons 390 that demonstrate impaired innate immune function can be immunomodulated to rescue or restore 391 function in vitro. Taken together with the observation that local BALF levels of vitamin D are 392 severely deficient in HIV+ persons, the current finding that exogenous 1,25D 3 partially rescued 393 the impaired innate macrophage host defense response in vitro suggests a potential therapeutic 394 role for 1,25D3 supplementation in HIV+ persons at risk for MTb disease. This study provides 395 the rationale to pursue additional in vitro investigations to allow the design of appropriate 396 clinical trials to define the role of exogenous vitamin D as a preventive or therapeutic adjuvant 397 for MTb infection, particularly in highly susceptible HIV+ persons. 398 Acknowledgements 399
The authors thank all volunteers who consented to research bronchoscopy. We thank Elizabeth 400
Vassar-Sternburg, Kristin Linnell, Ann Hougland, Xiomarra Guerra, Johanna Leary, Cynthia 401
Peguero, Jose Munguia and the BIDMC West Procedure Center staff for technical assistance 402
with research bronchoscopies. This work was supported by NIH T32-HL007118-33, NIH R01 403
HL063655 (H.K.), K08AI064014 (N.R.P), ALA Biomedical Research Grant (N.R.P). 404
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406 1. Anandaiah, A., K. Dheda, J. Keane, H. Koziel, D. A. Moore, and N. R. Patel. 2011. Novel 407 developments in the epidemic of human immunodeficiency virus and tuberculosis coinfection. 408 Am. J. Respir. Crit. Care Med. 183:987-997. doi: 10.1164/rccm.201008-1246CI. 409 2. Baldwin, A. S.,Jr. 1996. The NF-kappa B and I kappa B proteins: new discoveries and 410 insights. Annu. Rev. Immunol. 14:649-683. 411 3. Buhl, R., H. A. Jaffe, K. J. Holroyd, Z. Borok, J. H. Roum, A. Mastrangeli, F. B. Wells, 412 Download 226.29 Kb. Do'stlaringiz bilan baham: 
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