Franciska Southan1,2, Katherine Quinlivan1,2, Haixia Yang1,2,3, Diane R. Bielenberg4, Susanne I. Wells5, Charles N. Serhan6, Dipak Panigrahy1,2 1Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. 2Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. 3College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, 100083, China. 4Vascular Biology Program, Boston Children’s Hospital, Harvard Medical School, Boston, MA. 5Division of Oncology, Cancer and Blood Diseases Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH. 6Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA. Fanconi anemia (FA) is a condition distinguished by early-onset solid tumors, showing resistance to conventional cancer treatments such as chemotherapy and radiation. A major feature of FA is chronic inflammation, though the mechanisms governing the emergence and resolution of inflammation in FA remain poorly understood. We hypothesized that dysregulated resolution metabolomes and a systemic pro-inflammatory eicosanoid storm of inflammation-initiating mediators control FA tumor progression. To investigate we utilized a new murine model of transplantable FA tumors, focusing on head and neck squamous cell carcinoma (HNSCC) lacking the Fancc gene. We found that the advancement of Fancc-deficient HNSCC disrupts the resolution of inflammation by unbalancing specialized pro-resolving lipid mediators (SPMs) and eicosanoids. Tumor-bearing mice lacking Fancc demonstrated an increase in eicosanoids, notably leukotriene B 4 (LTB 4) and thromboxane B 2 (TXB 2 ) in the spleen alongside a 113% prostaglandin E metabolite (PGEM), a marker of PGE 2 production, in plasma compared to non-tumor-bearing (NT) mice. The non-tumor-bearing mice exhibit a decline in SPM/LTB 4 ratio, including RvD1/LTB 4, RvD2/LTB 4 , and MaR1/LTB 4 compared to NT mice. The activity of the immune checkpoint blockade in tumors deficient in mismatch repair, including head and neck cancers, indicates a relevance for immunotherapy in FA-induced cancers. Notably, individuals with FA maintain preserved levels of absolute T cells and CD4+T cell function. In a novel murine FA tumor model, we combined stimulation of the resolution of inflammation (i.e., treatment with RvDs) with immunotherapy. While treatment with RvD4, RvD5, or anti-PD1 immunotherapy alone delayed tumor growth compared to control at treatment day 20, monotherapy failed to prevent tumor escape by treatment day 30. Remarkably, combining RvD4 or RvD5 with anti-PD1 immunotherapy resulted in sustained regression of Fancc-/-tumors. Furthermore, RvD4 and/or anti-PD1 immunotherapy inhibit the growth of orthotopic Fancc-/- tumors compared to control. The coaction of resolvins and immune checkpoint blockade resulted in Fancc-/- tumor regression through mechanisms such as macrophage phagocytosis of apoptotic debris, counter-regulation of pro-angiogenic cytokines, and the inhibition of angiogenesis. By antagonizing Triggering Receptor Expressed on Myeloid cells-2 (TREM2) in conjunction with resolvins and immunotherapy, we restored SPM/eicosanoid ratios in Fancc-/- tumor-bearing mice to levels observed pre-cancer. SPMs and eicosanoids, therefore, present as potential early biomarkers and biological targets in FA-induced cancer progression. The stimulation of inflammation resolution via pro-resolution lipid mediators to bolster immunotherapy represents an innovative host-centric therapeutic approach, aimed at preventing FA-induced cancer progression through debris clearance and cytokine suppression. These results underscore the significance of targeting inflammation resolution via resolvins to enhance immunotherapy in the prevention and reversal of FA-induced cancer progression.

Th17 cells play an important role in autoimmune diseases, but the mechanisms regulating their migration into inflammatory tissues are not fully understood. We have previously reported that the discoidin domain receptor 1 (DDR1) enhances the migration of human polarized Th17 cells into collagen gels. In this study, we examined the role of DDR1 in arthritis. We found that human pathogenic Th17 cells (pTh17) also express and use DDR1 to migrate into collagen gels. The DDR1 kinase inhibitor (7rh) reduced DDR1 activation and the migration pTh17 cells into collagen gels. The effect of 7rh was confirmed by expressing the DDR1 dominant-negative form in human Th17 cells. We then examined the role of DDR1 in the mouse model of collagen-induced arthritis (CIA). Treatment of CIA mice with 7rh reduced the severity of arthritis, synovial inflammation and cartilage destruction. Blocking DDR1 also reduced Th17 cell infiltration into the joints, reduced proinflammatory cytokines and increased IL-10 cytokine levels suggesting that DDR1 is an important pathogenic pathway in arthritis. Our results indicate that DDR1 inhibition could reduce inflammation and tissue damage in collagen-rich tissues, like arthritic joints, and may therefore represent a new therapeutic target in autoimmune arthritis.

Specialized pro-resolving mediators (SPMs) including protectins, resolvins and maresins are recognized as important players in resolution of inflammation, and their therapeutic potential has been highlighted in many different models of inflammation related diseases. However, their translation into potent pharmaceutical drugs still encounters important hurdles including prohibitive cost of production, long chemical synthesis routes, patentability as natural product class, chemical sensitivity/instability, and sub-optimal physicochemical properties. In order to favor their emergence as valuable pharmaceutical class of compounds, we designed a novel strategy to simplify their chemical syntheses as well as to increase their related molecular diversity. Indeed, we have developed a molecular platform of simplified mimics of SPMs that structurally conserved the configuration of the central allylic trienic system (either E,Z,E; Z,E,E and E,E,E), but in varying their side chains. Protectin analogues with variable substituent groups were synthesized in 10-12 chemical steps to illustrate the viability of the approach. Interestingly, among the 40 protectin analogs synthesized, higher anti-inflammatory activities than their corresponding natural protectins (protectin DX and D1) were obtained in lipopolysaccharide (LPS) stimulated J744 macrophages, as well as enhanced antiviral activities on influenza and Sars-Cov-2 viruses. Some of these protectins analogues also showed promising preliminary pharmacokinetic profiles. These initial results reveal the potential of such structurally simplified mimics of SPMs as a valuable strategy towards their translation as future drug candidates.

Activated neutrophils expel neutrophil extracellular traps (NET), DNA/proteins fibers. Described as anti-microbial, NET can become immunogenic. NET formation is increased in rheumatoid arthritis (RA). We have previously shown that NET are pro-inflammatory on macrophages. We suggest that NET act as a DAMP and induce polyclonal B-cell activation, independently of antigen specificity. This mechanism may be amplified in RA patients. Here, we determined whether NET directly activate B lymphocytes, even non-autoimmune and non-memory B cells. Methods. Blood neutrophils/PBMC were isolated by density centrifugation from healthy donors (HD)/RA patients. Total or naïve B lymphocytes were purified by magnetic sorting. NET were induced in vitro by PMA or ionomycin. B lymphocytes were cultured in the presence/absence of HD/RA NET or LPS/CpG-oligonucleotide. Similar experiments were performed with cells from wild-type and TLR9-deficient mice. Cell purity/phenotype/activation were estimated by flow cytometry. Cytokine/IgG secretion/production were measured by ELISA/flow cytometry. Functional consequences of NET-activated B cells were analyzed on neutrophils/T cells by measuring migration, ROS/cytokine production, proliferation. The pathway triggered by NET was analyzed by RNA-seq. Results. NET induced a dose-dependent up-regulation of HLA-DR/co-stimulatory molecules by B lymphocytes both in HD and RA patients and both in total or naïve B lymphocytes. NET-activated B cells proliferate and secreted IL-8, IL-6, TNF, total IgG but not IL-10. The extent of B-cell activation suggests an antigen-independent polyclonal activation. Both HD and RA NET activate B cells, but RA B cells display a stronger response correlated with disease activity. B-cell responses depended on the NET-inducing stimulus. RNA-sequencing revealed NET-mediated up-regulation of RA pro-inflammatory genes and down-regulation of anti-inflammatory genes. Normal and TLR9-deficient B cells respond to NET. Finally, NET-activated B cells trigger neutrophil ROS production/recruitment and T-cell activation. Conclusions. NET directly trigger polyclonal B cell activation, even in naïve B lymphocytes, in a TLR9-independent manner, leading to an activated phenotype, a pro-inflammatory cytokine profile and neutrophil/T-cell activation. This mechanism is amplified in RA patients and may be pathogenic.