With the warmer season in full swing in Wisconsin, the grass needs mowing and the weeds need pulling. As a consequence, I am outside in the sun, synthesizing Vitamin D (and watching my freckles multiply). The benefits of this vitamin have been discussed in the news (e.g., may help prevent cancer, maintains a healthy working brain) while informing us most people lack adequate levels. A recent PLoS Pathogens article intrigued me because the researchers examined the role Vitamin D played in mitigating human immunodeficiency virus type-1 (HIV) and Mycobacterium tuberculosis coinfection in macrophages.
Why were Campbell and Spector interested in Vitamin D’s effect on HIV-1 and M. tuberculosis? First, they had previously shown that the active form of Vitamin D, 1α,25-dihydroxycholecalciferol (1,25D3), could inhibit HIV-1 replication by inducing autophagy in macrophages. This meant that the macrophages break down their own cell components. Autophagosomes (a vesicle) form around a subcellular organelle or microbe, fuse with a lysosome (an organelle containing acidic enzymes called hydrolases) and degrade the ingested items. Second, many people infected with HIV-1 are also infected with M. tuberculosis, and other research cited in the PLoS Pathogens article has implicated insufficient Vitamin D levels with increased susceptibility to M. tuberculosis, the causative agent of tuberculosis. Third, research on HIV-1 and M. tuberculosis has shown these infectious agents can reduce the number of autophagosomes and reduce the number of lysosomes in a cell, respectively. Thus, if Vitamin D could induce autophagy, it may counteract the decrease induced by HIV-1 and M. tuberculosis infection.
First the authors assessed how well physiological concentrations of the active form of Vitamin D 1,25D3 inhibited HIV-1 replication in macrophages by pretreating the cells with 1,25D3 then infecting them. The amount found in healthy individuals (100pmol/L) was able to reduce HIV-1 replication by 82% after 7 days. Half that amount showed 68% HIV-1 inhibition after 7 days while 200pmol/L showed greater inhibition HIV-1 replication than 100pmol/L, demonstrating the effect is dependent on concentration. Even when HIV-1 and M. tuberculosis coinfected macrophages pretreated with 1,25D3, HIV-1 replication was reduced.
Then Campbell and Spector tested how M. tuberculosis infection was affected by macrophage pretreatment with 1,25D3. Again, there was a dose-dependent growth inhibition seen at 50, 100 and 200pmol/L, and HIV-1 coinfection did not change these results. Thus whether macrophages were infected by HIV-1, M. tuberculosis or both, pretreatment with 1,25D3 reduced replication of the virus and bacteria both in the cells.
The reseachers then examined the induction of autophagy in macrophages coinfected with HIV-1 and M. tuberculosis. Using a molecular marker for autophagy induction, macrophages infected with HIV-1 or M. tuberculosis or both were treated with the healthy physiological concentration of 1,25D3 for 7 days. This marker indicated that autophagy had increased in the cells treated with 1,25D3. When staining for an indicator of autophagosome formation in the presence of 50–200pmol/L of 1,25D3, a similar dose-dependent increase was shown in infected macrophages. A third marker of autophagy flux also confirmed that autophagy increased in the infected macrophages. With this increased breakdown of cellular invaders, Campbell and Spector asked if there was cell death occurring with 1,25D3 treatment and confirmed there was no cytotoxic effects whether or not macrophages were uninfected or infected with HIV-1 or M. tuberculosis or both.
The work up to this point has shown that 1,25D3 pretreatment of macrophages can inhibit HIV-1 and M. tuberculosis infection of the cells and treatment of infected cells induces autophagy. To determine what might be happening in the cells, macrophages that were infected with HIV-1 or M. tuberculosis or both were either untreated or treated with 100pmol/L of 1,25D3 for 7 days and infection status and autophagosome formation assessed. The majority of untreated HIV-1-infected macrophages showed high levels of a marker for HIV-1 infection. The cells treated with 1,25D3 showed a much lower level of the HIV-1 infection marker, and those cells with the greatest reduction in the HIV-1 marker showed the greatest increase in the levels of autophagosome formation. For M. tuberculosis-infected macrophages, 75% of the untreated cells were infected with the mycobacterium with a third staining for autophagosome formation after 7 days. When the cells were exposed to 1,25D3, the level of infection was reduced and the autophagosome staining increased. For macrophages infected with both HIV-1 and M. tuberculosis, treatment with 1,25D3 increased autophagosome staining, reduced M. tuberculosis infection but the number of cells that were positive for HIV-1 were the same as untreated coinfected macrophages.
To understand where in the autophagy induction pathway 1,25D3 might exert its effect, Campbell and Spector used RNA interference (RNAi) to block various steps. Using RNAi to inhibit the formation of the complex that induces autophagosome formation, the marker of HIV-1 infection in the presence or absence of M. tuberculosis was reduced in the absence of 1,25D3, agreeing with the cited literature that HIV-1 uses autophagy machinery to replicate. When 1,25D3 was added to HIV-1-infected macrophages, the RNA silencing reversed much of the Vitamin D inhibitory effect on HIV-1 replication whether or not M. tuberculosis was also present. The effect of RNAi on the complex that induces autophagosome formation actually increased M. tuberculosis growth in the presence or absence of HIV-1 and attenuated the inhibitory effect of 1,25D3 on bacterial growth. Targeting a molecule directly involved with forming the autophagosome showed the same results as silencing the initiating complex for autophagosome formation: HIV-1 replication decreased and M. tuberculosis growth increased in the absence of 1,25D3 and the silenced molecule blocked the effect of 1,25D3 on autophagy.
In late stage autophagy, the autophagosome fuses with a lysosome to digest subcellular organelles and infectious agents. Using an inhibitor of the fusion step, there was little to no effect on HIV-1 replication in the absence or presence of M. tuberculosis, respectively. For M. tuberculosis, the inhibitor also had little to no effect on growth regardless of HIV-1 infection. Furthermore, the inhibitor also reduced the ability of 1,25D3 to increase autophagy. These same results were seen when an inhibitor of the acidic hydrolases in the lysosome was used.
Based on other research on 1,25D3, human cathelicidin microbial peptide (CAMP) is needed for inhibition of M. tuberculosis and induction of autophagy in macrophages. Therefore, CAMP was silenced and found to block 1,25D3 induction of CAMP when macrophages were coinfected with HIV-1 and M. tuberculosis, reduced 1,25D3-induced autophagy, eliminated 1,25D3-mediated antimicrobial activity and reversed 1,25D3 inhibition of HIV-1 replication in the presence of absence of M. tuberculosis.
Campbell and Spector showed that the active form of Vitamin D at a physiologically relevant concentration has a role in inhibiting the replication and growth of HIV-1 and M. tuberculosis in macrophages and this effect is mediated by autophagy. This is only a single research article using an in vitro method but does offer hope that Vitamin D may provide an supplemental treatment for physicians who manage patients who are HIV-1 positive and suffer from tuberculosis.
Campbell, G. and Spector, S. (2012). Vitamin D Inhibits Human Immunodeficiency Virus Type 1 and Mycobacterium tuberculosis Infection in Macrophages through the Induction of Autophagy PLoS Pathogens, 8 (5) DOI: 10.1371/journal.ppat.1002689
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One thoughtful comment
Wow! Autophagy is showing so much promise – especially with molecular inducers coming out like Dr. Levine’s Tat-Beclin1. It’s so cool to know that simple Vitamin D may have similar effects!
Vitamin D has so many effects. I think it’s because its so readily available from the sun so evolutionarily organisms took as much advantage of it as possible. Makes you wonder though — why be reliant on sunlight to change Vitamin D’s form? Why not just make an enzyme that converts it intrinsically? Hmm…