Systemic sclerosis (SSc) is characterized by skin/internal organ fibrosis, vasculopathy and autoimmunity. Chemokine (C-X-C motif) ligand 4 (CXCL4) is an SSc biomarker, predicting unfavorable prognosis and lung fibrosis. CXCL4 binds DNA/RNA and favors interferon (IFN)-α production by plasmacytoid dendritic cells (pDCs), contributing to the type I IFN (IFN-I) signature in SSc patients. However, whether CXCL4 is an autoantigen in SSc is unknown. Here, we show that at least half of SSc patients show consistent antibody reactivity to CXCL4. T-cell proliferation to CXCL4, tested in a limited number of patients, correlates with anti-CXCL4 antibody reactivity. Antibodies to CXCL4 mostly correlate with circulating IFN-α levels and are significantly higher in patients with lung fibrosis in two independent SSc cohorts. Antibodies to CXCL4 implement the CXCL4–DNA complex’s effect on IFN-α production by pDCs; CXCL4–DNA/RNA complexes stimulate purified human B-cells to become antibody-secreting plasma cells in vitro. These data indicate that CXCL4 is indeed an autoantigen in SSc and suggest that CXCL4, and CXCL4-specific autoantibodies, can fuel a harmful loop: CXCL4–DNA/RNA complexes induce IFN-α in pDCs and direct B-cell stimulation, including the secretion of anti-CXCL4 antibodies. Anti-CXCL4 antibodies may further increase pDC stimulation and IFN-α release in vivo, creating a vicious cycle which sustains the SSc IFN-I signature and general inflammation.

On the basis of previous studies demonstrating that a breach of the colonic epithelial barrier is associated with a microbiota-dependent increase in lamina propria (LP) regulatory cells, we investigated if the lack of spontaneous intestinal inflammation observed in nucleotide-binding oligomerization domain 2 (Nod2)−/− mice was due to enhanced intestinal regulatory function. We found that the LP CD4+ T-cell population of Nod2−/− mice contains an increased percentage of CD4+ regulatory T cells bearing transforming growth factor -β/latency peptide (LP CD4+LAP (latency-associated peptide) + T cells) both under baseline conditions and following an intentional breach of the colonic barrier induced by ethanol administration. In addition, we found that Nod2−/− mice manifest decreased severity of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-colitis and that TNBS-colitis in Nod2−/− or Nod2+/+ mice is ameliorated by adoptive transfer of LP cells from ethanol-treated mice before, but not after, depletion of LAP+ T cells. This increased regulatory T-cell response in Nod2−/− mice could explain why NOD2 polymorphisms in humans are not in themselves sufficient to establish inflammatory lesions.

Nucleotide-binding Oligomerization Domain-2 (NOD2) mutations are associated with an increased risk to develop Crohn’s Disease. In previous studies, we have shown that Nod2−/− mice manifest increased proportion of Lamina Propria (LP) CD4+ LAP+ Foxp3− regulatory cells, when compared with Nod2+/+ mice, while CD4+ Foxp3 + regulatory cells were not affected. Here, we investigated the Nod2 gut microbiota, by 16S rRNA pyrosequencing, at steady state and after TNBS-colitis induction in mice reared separately or in cohousing, correlating the microbial profiles with LP regulatory T cells proportion and tissue cytokines content. We found that enrichment of Rikenella and Alistipes (Rikenellaceae) in Nod2−/− mice at 8 weeks of age reared separately was associated with increased proportion of CD4+ LAP+ Foxp3− cells and less severe TNBS-colitis. In co-housed mice the acquisition of Rickenellaceae by Nod2+/+ mice was associated with increased CD4+ LAP+ Foxp3− proportion and less severe colitis. Severe colitis was associated with enrichment of gram-negative pathobionts (Escherichia and Enterococcus), while less severe colitis with protective bacteria (Barnesiella, Odoribacter and Clostridium IV). Environmental factors acting on genetic background with different outcomes according to their impact on microbiota, predispose in different ways to inflammation. These results open a new scenario for therapeutic attempt to re-establish eubiosis in Inflammatory Bowel Disease patients with NOD2 polymorphisms.

Background and Aims: Intestinal fibrosis is a common complication of inflammatory bowel diseases. Medical treatment of intestinal fibrosis is an unmet therapeutic need. CD147 overexpression can induce myofibroblast differentiation associated with extracellular matrix deposition, favouring the development of fibrosis. To understand whether CD147 may promote intestinal fibrosis, we analysed its expression and blocked its function by using its specific inhibitor AC-73 [3-{2-[([1,1’-biphenyl]-4-ylmethyl) amino]-1-hydroxyethyl} phenol] in the murine TNBS [trinitrobenzenesulfonic acid]-chronic colitis model associated with intestinal fibrosis. Methods: TNBS chronic colitis was induced by weekly intrarectal administration of escalating doses of TNBS. Ethanol-treated and untreated mice were used as controls. Separated groups of TNBS, ethanol-treated or untreated mice received AC-73 or vehicle administered intraperitoneally from day 21 to day 49. At day 49, mice were killed, and colons collected for histological analysis, protein and RNA extraction. CD147, α-SMA and activated TGF-β1 protein levels, CD147/ERK/STAT3 signalling pathway and autophagy were assessed by Western blot, collagen and inflammatory/fibrogenic cytokines mRNA tissue content by quantitative PCR. Results: In mice with chronic TNBS colitis, CD147 protein level increased during fibrosis development in colonic tissue, as compared to control mice. CD147 inhibition by AC-73 treatment reduced intestinal fibrosis, collagen and cytokine mRNA tissue content, without significant modulation of activated TGF-β1 protein tissue content. AC-73 inhibited CD147/ERK1/2 and STAT3 signalling pathway activation and induced autophagy. Conclusions: CD147 is a potential new target for controlling intestinal fibrosis and its inhibitor, AC-73, might represent a potential new anti-fibrotic therapeutic option in IBD.