Fibroblasts are essential for the development of cutaneous autoimmune diseases

Common autoimmune skin disorders are characterized
by regional onset.
For example, more than 80% of patients with vitiligo have a bilateral symmetrical distribution of onset, a type known as nonsegmental vitiligo (Figure 1)1,2
.
In vitiligo, autologous activated CD8+ T cells attack melanocytes, causing white patches
on the skin.
Between 0.
5 and 2 percent of the world's population suffers from the disease, but there is currently no FDA-approved treatment
.
Previous studies have shown that IFN-γ signaling is essential for skin depigmentation caused by CD8+ T cells3-6; However, the type of cells that mediate IFN-γ function is unknown
.
In addition, hypotheses for the regional onset of vitiligo include regional differences in the distribution of microbiota, differences in the expression of melanocyte antigens, and differences in neuropeptides released by nerve endings7-10
.
Elucidating the regulatory mechanisms that characterize the pathogenesis of autoimmune diseases can help develop effective treatment options
.

On December 16, 2021, Chen Ting's research team from the Beijing Institute of Biological Sciences/Institute of Biomedical Interdisciplinary Research of Tsinghua University published a research paper entitled Anatomically distinct fibroblast subsets determine skin autoimmune patterns in the journal Nature, through single-cell transcriptome sequencing, mouse models and a series of genetic experimental methods.
The important role of skin fibroblasts in vitiligo disease was revealed: fibroblasts are the only essential skin cells that can recruit and activate autologous active CD8+ T cells; The responsiveness of fibroblasts at different sites to IFN-γ determines their ability to recruit CD8+ T cells and determines the location preference
for vitiligo.

In order to study the characteristics of the symmetrical distribution of vitiligo at the cellular level, the authors first performed immunofluorescence staining on skin samples from vitiligo patients and found that CD8+ T cells were concentrated in large numbers at the junction of
lesions and non-lesions.
According to the distribution pattern of CD8+ T cells, it can be speculated that there is a recruitment mechanism during the occurrence of vitiligo disease, which coordinates the recruitment of CD8+ T cells at the lesion junction, so that CD8+ T cells are always at the forefront of the lesion skin, driving the gradual expansion
of the depigmented area.

Through single-cell transcriptome sequencing, the authors found that vitiligo patients had a higher proportion of killer CD8+ T cells in their skin than normal and expressed higher levels of IFNG.

GO analysis showed that the specific genes of melanocytes and fibroblasts in patients were enriched in the IFN-γ signaling pathway
.
Immunofluorescence staining of the downstream pSTAT1 signal of IFN-γ found that more than 80% of the pSTAT1+ cells in the skin of vitiligo patients were fibroblasts, indicating that fibroblasts are the most important cell type
that responds to IFN-γ signals in vitiligo patients.

To further investigate the regulatory mechanisms of vitiligo progression, the authors established a mouse model
of vitiligo.
After skin inoculation of B16F10 cells and intraperitoneal injection of anti-CD4 anti-CD4 antibody, C57 mice showed similar manifestations to vitiligo patients, including epidermal decolorization, CD8+ T cell aggregation infiltration, and melanocyte loss
.
The study found that after the same treatment, the infiltration of CD8+ T cells was significantly reduced compared with WT mice in transgenic mice with IFN-γ signaling receptor knockout (Ifngr1 KO), and there was no melanocyte deletion
in the epidermis.
This result suggests that cells that respond to IFN-γ signaling are critical
for the development and progression of vitiligo.

So, which cell type plays a decisive role? And how does it function? Chen Ting's team next used a variety of experimental methods to prove that in vitiligo disease, fibroblasts regulate the recruitment of CD8+ T cells through CXCL9 and CXCL10 (Figure 2).

First, the authors used the Cre-loxP system to specifically knock out Ifngr1
in each of the six cell types.
It was found that fibroblast-specific knockout of IFNGR1 effectively prevented vitiligo
.
The number of CD8+ T cells infiltrated by the epidermis of mice was significantly reduced, and the number of melanocytes was not affected, nor did it cause epidermal decolorization.
Other conditional knockout mice developed a vitiligo phenotype
.
Subsequently, the authors found through fibroblast transplantation experiments that in a local environment of the skin that did not respond to IFN-γ at all, only foreign fibroblasts that could respond to IFN-γ were sufficient to mediate the recruitment and aggregation
of local CD8+ T cells.
Third, Transwell experiments found that fibroblasts recruit activated CD8+ T cells
by secreting factors.
Compared with normal fibroblasts, chemokines CXCL9 and CXCL10 were upregulated
in vitiligo patients and vitiligo model mouse fibroblasts.
So, the researchers used lentiviral to knock down the Cxcl9 or Cxcl10 genes in dermal fibroblasts in WT mice, and found that fibroblasts that knocked down chemokines lost the ability
to recruit CD8+ T cells.

Fibroblast presence site heterogeneity11, fibroblasts in different parts of the mouse regulated the regeneration of hair follicles through the Hoxc gene regulation Wnt signaling pathway12
.
However, the regulation of immune responses by fibroblasts in different parts has not been studied
.
Through the analysis of 2265 patients with non-segmental vitiligo, the researchers found that the frequency of vitiligo in different parts was very different, with the highest incidence of the back of the hand and chest, and the lowest incidence of the
palm and upper limbs.
At the same time, the expression profiles of fibroblasts in different parts after IFN-γ treatment were also different, and CXCL9 and CXCL10 were upregulated
higher on the back of the hand, chest and back.
Further correlation analysis also showed that fibroblasts in sites with high incidence also responded more
to IFN-γ.
In addition, the researchers also found this correlation
in mouse models of vitiligo.
After the vitiligo model was induced, the melanocytes in the back and abdominal hair follicles of the mice were completely lost, but the melanocytes on the back of the paw and palm were not affected
.
Moreover, the expression levels of Cxcl9 and Cxcl10 in mouse back and abdominal fibroblasts after IFN-γ treatment were higher
than those on the back of the claw and palm.
The authors transplanted fibroblasts from the back and paw dorsal of WT mice onto Ifngr1 KO mice, and found that fibroblasts from the back of WT mice were more
capable of recruiting CD8+ T cells.
These experiments show that the responsiveness of fibroblasts at different sites to IFN-γ determines their ability to recruit CD8+ T cells and determines the location preference for vitiligo (Figure 3).

In summary, the study is the first to find that fibroblasts are essential for the occurrence of cutaneous autoimmune diseases; At the same time, there are regional differences in the regulation of autoimmune diseases, and the degree of response of fibroblasts in different parts to IFN-γ leads to different
ability to recruit T cells.
Fibroblasts not only exist in the skin, almost all organs have fibroblasts, and this study also provides reference
for the study of the mechanism of autoimmune diseases in other organs.

Professor Chen Ting of Beijing Institute of Biological Sciences and Professor Chang Jianmin of Beijing Hospital are the co-corresponding authors
of the paper.
Xu Zijian and Chen Daoming of the Beijing Institute of Biological Sciences are co-first authors
.
Other authors of the paper include Associate Researcher Hu Yucheng of Capital Normal University, Researcher Jiang Kaiju, Huang Huanwei, Du Yingxue, Wu Wenbo and Sui Jianhua from Beijing Institute of Biological Sciences, Dr.
Wang Wenhui and Dr.
Zhang Long from Peking University Third Hospital, Dr.
Li Shuli and Professor Li Chunying from Xijing Hospital, and Professor
Yang Yong from the Institute of Dermatology, Chinese Academy of Medical Sciences.
The work was completed
at the Beijing Institute of Biological Sciences.

Links to papers: https://doi.
org/10.
1038/s41586-021-04221-8

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