Giovanni Bernardini

Seed International Research Project (Last year report) NK CELL-BASED REGULATION OF MESENCHYMAL STEM CELL DIFFERENTIATION IN EXPERIMENTAL MODELS OF BONE FORMATION AND CARTILAGE DEGENERATION

GIOVANNI BERNARDINI Department of Molecular Medicine giovanni.bernardini@uniroma1.it

PETYA DIMITROVA Stephan Angeloff Institute of Microbiology petya_dimitrova@web.de

RESEARCH AREA: INFLAMMATION AND IMMUNITY

Our previous evidence show that NK cells and neutrophils are among the first cells that infiltrate the inflamed joint and exert a pathogenic role in a mouse model of collagenaseinduced experimental osteoarthritis (CIOA) (Benigni et al. J. Immunol. Vol 198, year 2017). We also showed that CXCL10/CXCR3 chemokine/chemokine receptor axis is crucial for disease establishment and progression, being CXCL10 increased in synovial fluid of CIOA mice and CXCR3-/- mice protected from disease development. CXCR3-/- mice maintain intact cartilage and bone despite the injection of degradative collagenase at the knee joint, suggesting that intrinsic abnormalities of cartilage/bone tissue formation and/or that impaired recruitment/activation of immune cells have a protective effect in CXCR3-/- mice. In support of the second hypothesis, during the project we found that osteoarthritic CXCR3-/- mice display reduced NK cell synovial infiltration. In addition, synovial fluids derived from OA mice promoted NK cell chemotaxis in in vitro migration assays and this was blocked by CXCL10 neutralizing antibodies. Nevertheless, Cxcr3+/+ and Cxcr3-/- NK cells are similarly recruited to the inflamed synovia, as demonstrated by competitive adoptive transfer experiments in vivo, thus suggesting that others CXCR3+ cells can indirectly participate in NK cell synovia infiltration. In order to investigate the mechanisms regulating NK cell contribution to disease progression and the possible role of CXCR3, our research activity has been focused on the analysis of the ability of NK cells to alter mesenchymal stem cell (MSC)s chondrogenic/osteogenic capacity. Thus, we investigated the role of CXCR3/CXCL10 axis in NK cell accumulation and activation in the joints during osteoarthritis and how CXCR3 regulates the process of bone/cartilage regeneration. In this way, we expected to determine the role of CXCR3 in NK cell dependent and independent regulation of MSC function, We further investigated the possible mechanism of NK cell recruitment by analysing the effect of CXCL10 on NK cell responsiveness to the chemokine CXCL12, a factor implicated in cartilage matrix degradation during osteoarthritis development.

Nevertheless, we found that in vitro CXCR3 antagonism leads to promotion rather than inhibition of NK cell responsiveness to CXCL12, thus excluding an indirect role played by CXCL12. We found that CXCL10 contributes to NK cell-mediated cartilage degenerative role as demonstrated by increased chondrocyte killing capacity promoted by addition of CXCL10 to NK cell/chondrocyte co-culture. This may be regulated by CXCL10 induction of death receptors ligands on NK cells since we could not show an effect of CXCL10 on NK cell degranulation, and we are currently exploring this hypothesis, In a second part of the project, we determined the effect of NK cells and of CXCR3 on mesenchymal stromal (MSCs) cell differentiation. MSCs were isolated from the bone marrow of Cxcr3+/+ and Cxcr3-/- mice and expanded by long-term culture in vitro. The purity of MSCs was determined by FACS analysis: isolated cells were CD45-, were Sca1+CD44+, and variably expressed CD90. At first, we investigated if activated NK cells produce factors able to modulate MSC differentiation or survival. We found that MSC were killed by in vitro co-culture with NK cells, but we could not find any regulation by CXCL10. Primary murine MSCs express chemokine receptors, including CXCR3 and this may be associated to a role in regulation of MSC migration and/or differentiation capacity (Chamberlain et al., PlosOne 2008). Thus, we analyzed the direct role of CXCR3 on MSC differentiation capacity by comparing the osteogenic capacity of the Cxcr3+/+ versus Cxcr3-/- MSC upon 2 weeks culture with factors able to promote osteoblast differentiation. Mineralized matrix formation after osteogenic differentiation was determined by Van Kossa staining. Analysis revealed matrix mineral deposition in Cxcr3+/+ but not in Cxcr3-/- MSC cultures. Cxcr3+/+ MSCs cultured without osteogenic medium were used as control. ALP activity assay confirmed differentiation of Cxcr3+/+ but not in Cxcr3-/- MSCs into osteoblasts (positive for ALP activity). On the other hand, we were unable to detect chondrocyte differentiation markers in both types of cultured cells. Overall, our results indicate that CXCR3 has a delelterious role in osteoarthritis mainly attributable to a double effect in disease development: CXCR3/CXCL10 axis promotes NK cell recruitment and activation in synovia that may exacerbate cartilage damage by promoting chondrocyte death. In addition, Cxcr3 expression by MSC promotes osteogenic differentiation with a mechanism not yet determined, and this may participate to inhibition of joint repair process. Thus, therapeutic approaches aimed at antagonizing the CXCR3/CXCL10 axis to treat osteoarthritis may be beneficial by reducing synovial inflammation and by promoting a correct joint repair.

Publications (2021)

1: Russo E, Laffranchi M, Tomaipitinca L, Del Prete A, Santoni A, Sozzani S, Bernardini G. NK Cell Anti-Tumor Surveillance in a Myeloid Cell-Shaped Environment. Front Immunol. 2021 Dec 17;12:787116. doi: 10.3389/fimmu.2021.787116. IF 7.5 2: Mormino A, Cocozza G, Fontemaggi G, Valente S, Esposito V, Santoro A, Bernardini G, Santoni A, Fazi F, Mai A, Limatola C, Garofalo S. Histone- deacetylase 8 drives the immune response and the growth of glioma. Glia. 2021. 69(11):2682-2698. doi: 10.1002/glia.24065. IF: 7.4 3: Tomaipitinca L, Russo E, Bernardini G. NK cell surveillance of hematological malignancies. Therapeutic implications and regulation by chemokine receptors. Mol Aspects Med. 2021 Aug;80:100968. doi: 10.1016/j.mam.2021.100968. Epub 2021. IF: 14.2 4: Mormino A, Bernardini G, Cocozza G, Corbi N, Passananti C, Santoni A, Limatola C, Garofalo S. Enriched Environment Cues Suggest a New Strategy to Counteract Glioma: Engineered rAAV2-IL-15 Microglia Modulate the Tumor Microenvironment. Front Immunol. 2021 Sep 6;12:730128. doi: 10.3389/fimmu.2021.730128. IF 7.5

Research Group

Luana Tomaipitinca Post-Doc; Eleonora Russo PhD student