NIAID Study Reveals Differences in Immune Cell Signaling in Humans and Mice

A new study by NIAID researchers uncovers key differences in the roles of certain cell-signaling proteins in mouse and human immune cells. Because mice often are used as models of human disease in preclinical research, it is critical to understand how they may differ from humans at the cellular level.

The scientists found that, overall, signaling in macrophages—immune cells that patrol the bloodstream and tissues—was similar in both mice and humans. However, they uncovered species-specific roles for specific signaling proteins, particularly members of a protein family called IRAK, which has been implicated in human autoimmune diseases. The findings, published in the January 5, 2016, issue of Science Signaling, highlight potential limitations for the use of mouse models to investigate human diseases. In addition, they may help scientists identify optimal clinical targets for drug design.

TLR Signaling: A Key Component of Immune Defense

Macrophages are key players in innate immunity, the nonspecific defense mechanisms that occur immediately when circulating immune cells recognize a problem. Innate immune cells respond quickly and broadly when a problem arises, typically leading to inflammation.

Scanning electron micrograph of a macrophage.

Credit: NIAID

Like other types of innate immune cells, macrophages contain genetically encoded receptors called Toll-like receptors (TLRs), which recognize general pathogen-associated patterns. TLRs respond to a range of microbial patterns, inducing a complex signaling response within cells and mounting an immune defense. Scientists have gleaned an overall understanding of TLR signaling from studies of different cell types in different experimental contexts.

Much of the current understanding of TLR signaling pathways comes from studies in mice. However, scientists have noted differences between human patients deficient in TLR pathway components and corresponding mouse models, raising questions about whether mice provide good models for studies of human immune diseases. For example, some researchers have proposed that the failure of numerous anti-inflammatory drugs in human clinical trials may be due to the drugs targeting a signaling component that does not behave the same way in animal models and humans.

To help address these questions and provide a complete picture of TLR signaling in a single innate immune cell type, researchers led by Iain Fraser, Ph.D., chief of the Signaling Systems Unit in the NIAID Laboratory of Systems Biology, conducted a systematic comparison of the contributions of human and mouse TLR pathway components in macrophages.

Developing a Screening Technique

The NIAID researchers turned to RNA interference, a research tool that allows scientists to silence specific genes. Short interfering RNAs (siRNA) act at the genetic level to turn off production of targeted proteins. Researchers then can observe what happens in cells in the presence and absence of a certain protein.

Applying RNA interference to immune cells presents considerable technical challenges, such as devising ways to efficiently deliver siRNA into the cells while avoiding nonspecific cellular responses to the RNA. The NIAID team developed a strategy to overcome these challenges, and in April 2015, they described a cell system for siRNA screening of pathogen responses in both human and mouse macrophages.

Uncovering Differences in IRAK Protein Usage

In the current study, the researchers used their siRNA-based screening strategy to assess the responses of laboratory-grown human and mouse macrophages to stimulation with various pathogen-derived molecules (see video). Their screen focused on 126 protein components of human and mouse TLR signaling pathways.

While they found that the core components of the signaling pathways in mice and humans are similar overall, the screening also revealed some unexpected differences. In particular, the scientists noted differences in usage of proteins from the IRAK family in mouse and human macrophages. Variations in IRAK proteins have been linked to various human autoimmune diseases, and the proteins also are considered promising targets for development of anti-inflammatory drugs.

Specifically, the NIAID scientists noted that IRAK1 and IRAK2 have different functions in human and mouse TLR signaling. In addition, while IRAK4 is essential for signaling in macrophages from both species, the functions of the mouse and human proteins appeared to differ.

The screen revealed that TLR signaling in mouse macrophages depended primarily on IRAK4 and IRAK2, while IRAK1 played little to no role. In contrast, human macrophages showed a different pattern, in which TLR signaling was highly dependent on IRAK1, with lesser roles for IRAK2 and IRAK4. The researchers confirmed these screening results with follow-up studies in macrophages taken from bone marrow tissue and blood. They also found that introducing human IRAK4 into mouse cells deficient in IRAK4 did not restore TLR signaling, nor could mouse IRAK4 restore signaling in IRAK4-deficient human cells, suggesting that the mouse and human forms of IRAK4 have different functions.

Identification of these previously unappreciated differences in IRAK usage in mouse and human macrophages promises to help inform ongoing clinical efforts to target this protein family to regulate inflammation and develop therapies for autoimmune disorders.

Mouse macrophages respond to LPS, a component of gram-negative bacteria. A transcription factor protein from the NF-κB signaling pathway (in green) responds to LPS by moving from the cell cytosol into the cell nucleus to promote the expression of immune inflammatory genes. The expression of one such inflammatory gene, TNF-α (in red), is increased following the activation and accumulation of NF-κB in the macrophages’ nuclei. This 21-second video represents a 21-hour recording.

References

Sun J, Li N, Oh K-S, Dutta B, Vayttaden SJ, Lin B, Ebert TS, De Nardo D, Davis J, Bagirzadeh R, Lounsbury NW, Pasare C, Latz E, Hornung V, Fraser IDC. Comprehensive RNAi-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use. Science Signaling DOI: 10.1126/scisignal.aab2191 (2016).

Li N, Sun J, Benet ZL, Wang Z, Al-Khodor S, John SP, Lin B, Sung M-H, Fraser IDC. Development of a cell system for siRNA screening of pathogen responses in human and mouse macrophages. Scientific Reports DOI: 10.1038/srep09559 (2015).