PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PKcs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs.

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Monocytes and monocyte-derived cells are important players in the initiation, progression, and resolution of inflammatory skin reactions. As inflammation is a prerequisite for fibrosis development, we focused on the role of monocytes in cutaneous fibrosis, the clinical hallmark of patients suffering from systemic sclerosis. Investigating the function of monocytes in reactive oxygen specieseinduced dermal fibrosis, we observed that early monocyte depletion partially reduced disease severity. Low numbers of inflammatory Ly6C high monocytes, as well as inhibition of CCR2 and CCL2 in wild type animals by a specific L-RNA aptamer, mitigated disease parameters, indicating a pivotal role for CCR2 þ inflammatory monocytes and the CCR2/CCL2 axis in fibrosis development. Of note, mice lacking splenic reservoirs failed to recruit monocytes to the skin and developed less fibrosis. Furthermore, enforced monocyte conversion into noninflammatory, patrolling Ly6C low monocytes by a nuclear receptor Nur77eagonist also resulted in significantly impaired cutaneous inflammation and dermal fibrosis. Most evident, pronounced monocyte conversion in interferon stimulated gene 12edeficient mice with pronounced nuclear Nur77 signaling completely protected from dermal fibrosis. Our study shows that inflammatory monocytes that are recruited from splenic reservoirs play a key role in the development of skin fibrosis and can be therapeutically challenged by forced conversion via the Nur77/interferon stimulated gene 12 axis.

Mouse models resembling systemic sclerosis can be chemically induced by application of bleomycin or hypochloric acid (HOCl). To date, little is known about inflammatory cells and their potential role in scleroderma (Scl)-related fibrosis. Therefore, we compared both Scl models to define the early immune cell subsets in relation to fibrosis-related parameters. Both agents induced a significant increase in dermal thickness and collagen deposition after 4 weeks, as hallmarks of Scl. However, clinical skin thickness, densely packed, sirius red-stained collagen bundles and collagen cross-links were more pronounced in HOCl-induced Scl. In parallel, there was a significant upregulation of procollagen α1(I), α-SMA and TGF-β transcripts in HOCl animals, whereas IL-1β and MMP-13 mRNA levels were significantly increased in bleomycin-treated mice. Flow cytometric analysis of the Scl skin demonstrated an early cellular infiltrate containing mainly CD19 B cells, CD4 T cells, CD11c DC and CD11b myeloid cells, the latter ones being significantly more prominent after HOCl injection. Subanalysis revealed that Scl mice exhibited a significant increase of inflammatory myeloid CD11b Ly6C CD64 cells (HOCl>bleomycin). In particular, in the HOCl model, activated dermal macrophages (CCR2 MHCII ) and monocyte-derived DC (CCR2 MHCII ) predominated over less activated CD11b myeloid cells. In conclusion, the two models differ in certain aspects of the murine and human scleroderma but in the HOCl model, myeloid CD11b MHCII cells correlate with some fibrosis-related parameters. Therefore, analysis of both models is suggested to cover a comprehensive profile of Scl symptoms but with focus on the HOCl model when the role of early myeloid immune cells will be evaluated.

We show that Scurfy mice show a predominant AC-5 ANA pattern typical for mixed connective tissue disease as in scleroderma. The autoimmune inflammation in scurfy skin mainly consists of CD4 T cells with Th2 differentiation and alternatively-activated (M2) macrophages as it is found in scleroderma with advanced fibrosis.