Researchers at the Program in Cellular and Molecular Medicine and the Immune Disease Institute at Children's Hospital Boston (PCMM/IDI) have uncovered a novel role for the transcription factor NFAT5: it is a major mediator of toll-like receptor (TLR) signaling resulting in gene transcription, and via this pathway it plays a key role in driving the stimulation of HIV viral load by TB co-infection. TB, which is caused by Mycobacterium tuberculosis (MTb), is the leading cause of death among HIV-infected persons.
Reporting online in PLOS Pathogens, researchers in the laboratory of Dr. Anne Goldfeld have not only determined that NFAT5 plays a crucial role in direct regulation of HIV-1 replication by MTb, but they also demonstrate for the first time a general role for NFAT5 in control of gene expression, mediated by MTb and the TLR signal transduction pathway. These findings help explain the mechanism by which TB elevates HIV viral load and the secondary devastating effects of HIV/TB co-infection, including high mortality. Eventually these studies may also lead to new therapeutic targets for treatment of HIV-1 and possibly other diseases.
Understanding the transcriptional regulation of genes is a long-standing interest of the Goldfeld lab, with the regulation of transcription and replication of HIV a main research focus. Dr. Goldfeld explains: "We're very interested in how TB infection manipulates the host environment to result in different outcomes of HIV biology. If you can figure that out, you can figure out ways to intervene."
TB co-infection is known to speed up HIV-1 replication and disease progression through both innate and adaptive host immune responses. First author Dr. Shahin Ranjbar had previously shown that different TB strains drive HIV expression in different patterns, an effect she showed to be produced through differential cytokine responses.
A key feature of the current work was the creation of a lab model by Dr. Ranjbar that recapitulates co-infection using currently circulating isolates of both TB and HIV, i.e., bacteria and viruses gathered in field work in areas where co-infection is a huge problem - rather than lab-adapted strains. These isolates were used to infect either monocytes or the entire peripheral blood mononuclear fraction from clinical samples, not laboratory cell lines. All those decisions were part of a strategy designed to mimic as closely as possible in vivo conditions in an ex vivo system.
The results of the study unmasked an important but novel player in the drama of HIV and TB. Ten years ago, the main thing known about the transcription factor NFAT5 (nuclear factor of activated T cells 5) was that it was induced by hyperosmotic shock. Then in 2006 Dr. Ranjbar showed for the first time that NFAT5 is a key factor in HIV replication in non-activated cells, particularly in monocytes. That work employed early small interfering RNA (siRNA) technology, which stops the expression of any gene of interest.
But that earlier project provided another clue to the central role of NFAT5: its consensus binding site was more highly conserved than any other regulatory sequence in the HIV's control center for gene expression, the long terminal region (LTR). In other words, NFAT5 had been so important during the virus' evolutionary process that it remained intact in all HIV's variants: different subtypes of HIV-1, and its close relatives, HIV-2, SIV, etc. Dr. Ranjbar took what had been learned about the sequences involved in constitutive HIV regulation in macrophages and applied it to macrophages co-infected with TB.
The Goldfeld team knew that TB relies upon Toll-like receptor 2 (TLR2) to accomplish gene expression of both cytokines and HIV. TLR2 plays a fundamental role in pathogen recognition and activation of innate immunity. The lab first showed that when you directly stimulate TLR2, it upregulates NFAT5. Dr. Ranjbar then interrupted the signal transduction pathway from TLR2 to NFAT5, using another new and powerful tool, short hairpin RNA (shRNA) and showed that the TLR2 signaling pathway results in NFAT5 transcription.
Thus, the Goldfeld lab has shown for the first time that expression of NFAT5 is strongly induced by TB, which is a Toll-like receptor (TLR) ligand, and through mutagenesis of circulating isolates and the construction of infectious molecular clones they showed that efficient induction of HIV replication by TB requires an intact NFAT5 binding site in the HIV promoter. Furthermore, NFAT5 gene expression induced by MTb in human monocytes could be silenced using RNAi of key components of the TLR pathway.
Dr. Goldfeld notes that the previous understanding of possible targets for interventions in HIV transcription was limited to other cellular transcription factors such as NF kappa B. "But we were able to show that perfectly intact NF kappa B sites in the HIV LTR could not 'rescue' the virus when NFAT5 sites in the HIV LTR were mutated."
Dr. Ranjbar and her fellow Goldfeld lab member Dr. Luke Jasenosky managed the very difficult task of creating infectious molecular clones of HIV with specific mutations at the NFAT5 site, then compared these to their wild-type HIV counterparts. Dr. Goldfeld further explains the complexity of the work published in PLOS Pathogens. "One hard part was working under containment conditions with the contagious and serious infectious agents, one respiratory and the other bloodborne. The molecular biology here was also quite complex."
These discoveries have provided new directions for the Goldfeld lab in their work with HIV/TB co-infection. Topics already under way or contemplated for the future include relating immunological profiles with survival rates in patients who are co-infected with HIV and TB or HIV alone; gaining a better understanding of extreme inflammatory reactions in response to HIV treatment in TB/HIV co-infected patients; and examining how the chromatin environment and methylation state of surrounding histones affects HIV gene expression and replication. In turn, that research will also be brought to bear on HIV in terms of viral latency and new infection - with TB as a co-factor.
Dr. Goldfeld sums it up: "What's very satisfying is when all the pieces fit together, after you've started with a big question mark about something previously unknown. When we mutated the NFAT5 site, we crippled the virus. When we blocked NFAT5 it crippled the virus. The whole thing as a story just makes beautiful sense."
Ranjbar S, Jasenosky LD, Chow N, Goldfeld AE. Regulation of Mycobacterium tuberculosis-dependent HIV-1 transcription reveals a new role for NFAT5 in the toll-like receptor pathway. PLoS Pathog. 2012;8(4):e1002620. Epub 2012 Apr 5.
