These findings are reported Dec. 16 in the advanced online edition of the journal Nature. The research was a collaboration between the 91̽»¨ and the Seattle Biomedical Research Institute.

Dr. Lalita Ramakrishan, who studies how TB evades the body’s immune system and manipulates the body’s defenses for its own ends, is the senior author. She is a 91̽»¨professor of microbiology, medicine and immunology. The lead author is C.J. Cambier of the 91̽»¨Department of Immunology.

Ramakrishnan noted that the recent study suggests an explanation for the longstanding observation that tuberculosis infections begin in the comparatively sterile lower lungs. In the upper respiratory tract, resident microbes and inhaled microbes of a variety of species signal their presence.

These tip-offs alert and attract many infection-fighting cells to the upper airways. The presence of other microbes in the upper airway may thereby help to keep TB infections at bay by creating a hostile environment.

Their presence may explain why TB is less contagious than diseases caused by several other respiratory pathogens.

To produce an illness, TB bacteria must sneak through this well-patrolled area and head for parts of the lungs where fewer microbiocidal cells are policing.

Like most other bacteria, TB pathogens have telltale molecular patterns that should activate an immune response. However, TB pathogens have evolved mechanisms to circumvent tripping the alarm. Almost like home intruders wearing a stocking over their faces, the TB pathogens produce particular types of fatty substances, or lipids, on their cell surfaces.

These lipids, abbreviated as PDIM, are already known to be associated with bacterial virulence. The researchers showed that PDIM lipids function by masking the underlying molecular patterns that would reveal their dangerous nature to infection-fighting cells.

At the same time, a related lipid – called PGL – on the bacterium’s cell surface promotes the recruitment of clean-up cells that engulf but don’t kill the TB pathogens. Instead, they take them across the lung lining, deep into the lung tissue where the bacteria can establish an infection.

The TB pathogens then use the other lipid molecule, PGL, to co-opt a host chemical pathway that triggers the recruitment of the permissive macrophages.

The present study expands on earlier work in the Ramakrishan and collaborative labs, which helped describe the strategies by which TB pathogens manipulate host pathways for their own purposes after they enter certain host cells.

These include the secretion of proteins that expand the niche for TB by recruiting macrophages to the early lung tubercles characteristic of the disease. The present study describes earlier stages in infection, when the pathogens first come in contact with their potential host at the surface of the lung lining.

“The current study suggests the manner in which the TB pathogens manipulate recruitment of the first responding macrophages to gain access to their preferred niche,” the researchers noted.

“The choreographed entry involves two related TB cell lipids acting in concert to avoid one host pathway while inducing another,” they wrote. The findings link the previously known, absolutely essential virulence factor on the surface of TB cells, PDIM, to the evasion of immune cell detection. On the other hand, PGL is not required on the surface of TB cells for them to infect the body.

Ramakrishnan noted that globally, a lot of samples of TB taken from infected patients do not have PGL. “However,” she and her research team noted, “the importance of PGL in mediating TB virulence or transmission is underscored by its presence in many of the W-Beijing strains” of TB which are starting to appear in more patient samples, and which have predominated in outbreaks in North America.

Ramakrishnan explains that their findings suggest how PGL may be important in increasing TB’s infectivity.

“The presence of PGL in ancestral strains of TB suggest it played an integral role in the evolution of TB infectivity,” the researchers noted. “TB is an ancient disease and the enhanced infectivity conferred by PGL may have been essential for most of its history before human crowding, with its increased opportunity for transmission, made it dispensable.”

The study findings, and previous work on TB, might also explain why smaller droplets of TB are more infectious than larger ones. Only the smaller droplets can make their way down into the lower airways. All it takes is 3 or fewer TB mycobacteria with PGL-producing ability to enter the lower lungs and start an infection.

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The other researchers on the study, in addition to Cambier and Ramkrishnan, were Kevin K. Takaki, David M. Tobin, and Christina L. Cosma, all of the 91̽»¨Department of Microbiology; Ryan Larson and Kevin N. Urdahl of the of the 91̽»¨Department of Immunology and the Seattle Biomedical Research Institute. Urdahl also is from the 91̽»¨Department of Pediatrics.

The research was supported by training and research grants from the National Science Foundation, American Lung Association, National Institutes of Health, and American Cancer Society. Tobin is an NIH New Innovator and Ramakrishnan is an NIH Pioneer.

Tumor necrosis factor – normally an infection-fighting substance produced by the body – can actually heighten susceptibility to tuberculosis if its levels are too high.

91̽»¨ TB researchers unravel this conundrum in a report this week in Cell.

Their study shows how excess production of this disease-cell destroyer at first acts as a TB germ killer. But later the opposite occurs: too much tumor necrosis factor encourages TB pathogens to multiply in the body.

In addition to figuring out some reasons behind this back-pedaling, the scientists learned that certain combinations of drugs already available for other conditions can curtail the shift from anti-TB to pro-TB.

The drug combination revealed in this study, the authors noted, “has the potential to revert some cases of hypersusceptibility to hyperresistance.”

The scientists were Francisco Jose Roca Soler, of the 91̽»¨Department of Microbiology, and Lalita Ramakrishnan, 91̽»¨professor of microbiology, medicine and immunology. A recipient of the National Institutes of Health Director’s Pioneer Award, Ramakrishnan is recognized for her work on how the TB pathogen and its hosts’ cells interact to cause disease.

These studies are conducted in zebrafish, an animal model for tuberculosis. The fish’s embryos and small fry are transparent. Researchers can see through their skin to observe their organs, tissues and cells and the internal appearance of some infections, for example, the bacterial cording of TB.

Roca and Ramakrishnan explained that TB had traditionally been thought of as a disease of failed immunity. However, more recent studies from their lab and other labs, both in zebrafish and in humans, have suggested that it also can result from too strong of a defensive inflammatory response.

“While tumor necrosis factor is a critical host defense against tuberculosis,” Roca and Ramakrishnan noted, “an excess of this factor is also implicated in the development of the disease in zebrafish and in humans.”

Variations in a specific location of the zebrafish genome can cause either too much or too little tumor necrosis factor to be produced, depending on the type of variation. In either case, deficiency or overabundance, zebrafish become prone to tuberculosis.

In both cases the scavenger cells, or macrophages, that are trying to clear away the TB pathogens by ingesting them, die and burst open. They are like torn vacuum cleaner bags spilling their dirty contents.

When the TB bacteria escape the confines of the scavenger cells, “they grow exuberantly in the extracellular environment,” Roca and Ramakrishnan said.

Researchers needed to work out the differences between TB susceptibility caused by too high or too low tumor necrosis factors because the distinction is vital to treatment decisions. Only patients whose genetics made them launch a pro-inflammatory response, benefited from steroid treatment, previous studies have shown. Steroids can increase the chance of death among TB patients with a weak inflammatory response.

In the present study, Roca and Ramakrishnan elucidated the molecular pathways by which too much tumor necrosis factor at first rapidly promotes macrophages to go after TB bacteria, and then turns around and forces the hard-working macrophages to die and expel their captives.

They found that both the microbiocidal activity, and the death of the macrophages, resulted from upping the production of reactive oxygen species by the mitochondria inside the macrophages. Mitochondria are the energy-generating power plants of living cells.

Tumor necrosis factor inside of infected macrophages induces reactive oxygen species from the mitochondria. These are the chemicals responsible for cell damage from oxidative stress.

Early on, reactive oxygen species can be beneficial. Initially their presence encourages the macrophages to destroy pathogens. As they accumulate, however, they promote self-harm.

Suddenly the macrophage is programmed to self-destruct. The reactive oxygen species carry out the death sentence by modulating a pathway for a substance called cyclophilin D, which sets the stage for the demolition of mitochondria.

Reactive oxygen species also play a role in acid sphingomyelinase-mediated ceramide production. This waxy substance occurs in cell membranes. One of its many roles is regulating signals for cell death.

The researchers were able to convert the high tumor necrosis factor state to become resistant to tuberculosis. They did so by genetically blockading both cyclophilin D and acid sphingomyelinase in previously susceptible zebrafish.

Similarly, they discovered that the drug combination of alisporivir, a cyclophilin D-inhibiting drug, and desipramine, an antidepressant that inactivates acid sphingomyelinase, also reverses susceptibility to TB in zebrafish prone to tumor necrosis factor excess.

Essentially, the experiments suggest that preventing cell death in TB infected macrophages can prolong their capacity to attack TB pathogens.

A longer-living army of macrophages, filled with the microbiocidal reactive oxygen species, will destroy the TB pathogens inside them and make the host highly resistant to tuberculosis.

Because excessive amounts of tumor necrosis factor are implicated in several inflammatory diseases such as rheumatoid arthritis, ankylosing spondylitis, sarcoidosis, and Crohn’s, the authors noted, “The findings may be useful for understanding diseases in addition to tuberculosis.”

Grants from the National Institutes of Health and the Northwest Research Center of Excellence for Biodefense and Emerging Diseases, and a postdoctoral fellowship from the educational ministry of Spain, funded this research project.

The Cell paper is titled, “Tumor necrosis factor dually mediates resistance and susceptibility to mycobacteria through induction of mitochondrial reactive oxygen species.”