Immunologists at Sun Yat-sen University (SYSU) in Guangzhou, China, have been the first to show that interferon-induced protein 35 (IFP35) family proteins promote neuroinflammation and multiple sclerosis (MS), as they reported in the August 2, 2021, edition of Proceedings of the National Academy of Sciences.

These findings not only improve the understanding of the complex pathogenesis of MS, but also provide new potential diagnostic and therapeutic targets for managing the chronic CNS disease.

MS is the major cause of nontraumatic disability in young adults, with pathological characteristics including demyelination, inflammatory cell infiltration and axon damage.

However, despite intensive research, the etiology and molecular mechanisms underlying MS initiation and progression remain unclear.

"The etiology of MS is known to be complex, since neurobiological diseases remain among the least well-known areas in biological sciences," study co-leader Yingfang Liu, professor in the School of Medicine at SYSU, told BioWorld Science.

A dysregulated immune system is apparently responsible for MS exacerbation, with multiple immune cells thought to damage myelin sheaths, oligodendrocytes, axons and neurons.

Following CNS entry, T cells mount an inflammatory response against myelin auto-antigens, with T-helper 1 (Th1) and Th17 cell cytokines and chemokines promoting microglial and macrophage activation.

Activated microglia produce multiple neurotoxic factors that facilitate neuroinflammation and pathogenic encephalomyelitis.

Cytokines and chemokines from activated microglia and T cells facilitate peripheral immune cell CNS recruitment, whereas Th2 cell- and T regulatory (Treg) cell-mediated immune responses are associated with reduced inflammation and beneficial clinical symptom effects in MS patients.

The experimental autoimmune encephalomyelitis (EAE) model is usually used to study molecular mechanisms underlying MS, since it has clinical and immunopathological similarities to those in MS patients.

Although several disease-modifying treatments are used clinically to alleviate symptoms, MS remains essentially incurable, necessitating elucidation of factors underlying T-cell and microglial activation to identify disease targets.

Inflammatory responses are exacerbated by endogenous proinflammatory damage-associated molecular pattern (DAMP) molecules, which are released by immune cells after cellular stress or tissue injury.

DAMPs promote activation of CNS and peripheral immune responses, while MS lesion cell debris is rich in DAMPs that may contribute to persistent inflammation, damaging neurological tissues during MS development.

IFP35 family proteins

IFP35 family proteins, including NMI (N-myc and STAT-interactor protein), have been shown to function as DAMPs to promote host inflammatory responses in sepsis and are involved in multiple other inflammatory diseases.

Levels of IFP35 expression have recently been correlated with progression and outcome of neuroinflammation in MS, but the mechanisms linking IFP35 proteins and MS are unclear.

Because extracellular IFP35 and NMI are known to activate macrophages as peripheral proinflammatory molecules, researchers led by Liu and Huanhuan Liang, a professor in the School of Pharmaceutical Sciences at SYSU, investigated their peripheral and CNS neuroinflammatory functions.

A clinical transcriptomic data analysis showed that "IFP35 family protein expression was upregulated to a significantly greater extent in gray matter lesion tissues from MS patients versus controls," said Liu, while in vitro studies established that IFP35 and NMI were released by multiple cells.

The researchers, who included Xizhong Jing, Yongjie Yao and other SYSU scientists, further demonstrated that IFP35 and NMI triggered NF-kappaB-dependent activation of microglia via the Toll-like receptor 4 (TLR4) signaling pathway.

"We used recombinant IFP35 family proteins to stimulate microglial activation and inflammatory cytokine release, while p50 and p65 subunits of NF-kappaB were also seen to migrate from cytoplasm to nucleus," noted Liu.

However, "when using a TLR4 inhibitor or TLR4-deficient microglia, the induction activity of recombinant proteins was almost lost, so we conclude that IFP35 and NMI triggered NF-kappaB-dependent activation of microglia via TLR4 signaling pathway," he explained.

"Since microglia are the major CNS immune cells, their abnormal activation is closely related to MS pathogenesis and our findings reveal that IFP35 family proteins are novel activators of microglia through TLR4/NF-kappaB pathway, which we expect to inspire further MS research."

Both IFP35 and NMI were shown to activate dendritic cells and promote naive T-cell differentiation into Th1 and Th17 cells.

This is important, "because MS is an autoimmune demyelinating disease in the CNS, with self-activated T cells attacking CNS myelin sheath being the main cause of the disease," Liu said.

"Th1 and Th17 cells are the predominant pathogenic T cell types in MS, while activation and differentiation of naive T cells to Th1 and Th17 cells require assistance of antigen-presenting dendritic cells," he explained. Therefore, "we propose that the IFP35 family proteins play important roles in the pathogenesis of MS."

Interestingly, in Nmi and Ifp35 genetic knockout (KO) mice created using CRISPR-Cas9 gene editing, administration of neutralizing antibodies against IFP35 alleviated immune cell infiltration and demyelination observed in the CNS, and reduced the severity of EAE.

"While the incidence of EAE was not significantly reduced in Nmi and Ifp35 KO mice, symptoms were noticeably improved at the peak stage 15-20 days after immunization," said Liu.

"Clinical scores were decreased by about 40% in KO mice or after antibody administration versus controls, while areas of immune cell infiltration and CNS demyelination were also visibly reduced."

Together, these findings reveal a previously unknown mechanism by which IFP35 family proteins facilitate T-cell and microglial overactivation and suggest ways to study MS pathogenesis.

Notably, "we found that IFP35 family proteins can be released by damaged or activated CNS cells, but they can also be secreted from MS lesions to cerebrospinal fluid and the circulation, identifying IFP35 and NMI as potential targets for the development of novel diagnostics for MS."