**1. Introduction**

Rheumatoid arthritis (RA) is a chronic, autoimmune disease, characterized by inflammation and progressive damage of synovial joints, when treated insufficiently [1]. With increasing knowledge about disease pathophysiology, new pharmaceutical strategies and compounds are available. After the application of conventional synthetic diseasemodifying antirheumatic drugs (csDMARDs), such as methotrexate (MTX) in late 1980s, and biological DMARDs since late 1990s, small-molecule Janus kinase inhibitors (JAKis), classified as targeted synthetic DMARDs, represent a new milestone in RA treatment [1–3]. Clinical studies with JAKi demonstrated similar efficacy and safety compared to biological DMARDs [4,5]. However, long-term data for JAKi covering several years are still missing.

Janus kinases (JAKs) are cytoplasmic tyrosine kinases comprising four different types of JAK enzymes in humans: JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2) [6]. While JAK1, JAK2, and TYK2 are expressed ubiquitously, JAK3 is predominantly detectable in hematopoietic tissue [7,8]. Upon extracellular ligand binding, JAKs associate as homo- or

**Citation:** Reddig, A.; Voss, L.; Guttek, K.; Roggenbuck, D.; Feist, E.; Reinhold, D. Impact of Different JAK Inhibitors and Methotrexate on Lymphocyte Proliferation and DNA Damage. *J. Clin. Med.* **2021**, *10*, 1431. https://doi.org/10.3390/jcm10071431

Academic Editor: Mitsuhiro Takeno

Received: 2 February 2021 Accepted: 29 March 2021 Published: 1 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

heterodimers with type I and type II cytokine receptors. Subsequently, JAK dimers become activated by auto- and transphosphorylation and phosphorylate the cytoplasmic tail of the cytokine receptor [6,8]. This induces the recruitment and binding of signaling molecules, such as the members of the signal transducer and activator of transcription (STAT) family (seven members: STAT1/2/3/4/5A/5B/6). After JAK-mediated STAT phosphorylation, dimerization, and activation, STAT dimers translocate to the nucleus where they act as transcription factors for multiple target genes, modulating, i. a., survival, proliferation, or differentiation of T lymphocytes [9,10]. More than 50 different cytokines and growth factors are known ligands of type I/II cytokine receptors. Depending on the cytoplasmic chains of the receptor, they are able to associate either with only one type of JAK enzyme or with different JAK isoforms. Hence, this creates a high degree of specificity regarding different JAK and STAT combinations [9–11].

Sufficient JAK-STAT signaling is essential in the regulation of immunological processes. Polymorphisms and loss- or gain-of-function mutations within this pathway are associated with immunodeficiency, autoimmune disease, and hematological malignancy [9,12]. Therefore, JAK-targeting agents represent a new class of immunomodulatory drugs [11]. After first approval of JAK inhibiting drugs for the treatment of neoplastic diseases, several JAKi are also authorized for the treatment of RA by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) [13,14]. The two approved first-generation JAK inhibitors tofacitinib (JAK3 > JAK1 > JAK2) and baricitinib (JAK1 > JAK2 > JAK3) are classified as pan-JAK inhibitors, targeting multiple JAK isoforms, but with different affinities. In contrast, the two second-generation JAKi upadacitinib and filgotinib (not yet approved by the FDA) show high selectivity for JAK1 and primarily inhibit its associated cytokine-receptors [10,15]. However, selectivity of JAKi towards specific JAK isoforms is not absolute and depends on dosage and cell type [16]. An overview of reported mean half-maximal inhibitory concentrations (IC50) obtained by enzymatic assays is provided in Table S1.

Although already approved for RA treatment, in vivo and in vitro head-to-head studies of all four JAKi are rare. Therefore, we evaluated the immunomodulatory and cytotoxic potential of tofacitinib, baricitinib, upadacitinib, and filgotinib on human PBMCs freshly isolated from healthy donors. For comparison with conventional synthetic DMARDs, samples treated with MTX were investigated in parallel. JAKi or MTX were either added directly to freshly PHA-stimulated PBMCs or 48 h after PBMC activation, to investigate their impact on preactivated T lymphocytes, as this might be more relevant regarding inflammatory conditions in vivo [17]. Compared to healthy controls, peripheral blood isolated from patients with active RA revealed an enhanced level of activated PBMCs, which may play a direct role in disease pathogenesis [18]. Furthermore, Kitanaga et al. stated constitutive activation of JAK/STAT signaling in PBMCs from patients with systemic sclerosis or RA [19].

JAK/STAT signaling is involved in regulation of multiple fundamental cellular processes. Additionally, there is increasing evidence suggesting that JAK/STAT signaling also modulates molecules involved in DNA damage response pathways [20–24]. To investigate the impact of JAKi on DNA double-strand break (DSB) formation and on repair of radiationinduced DNA damage we quantified nuclear foci stained by γH2AX or 53BP1 (p53-binding protein 1) antibodies. These markers have been described as sensitive molecular indicators for DNA DSBs [25–27].

The objective of the present in vitro study was to compare the immunomodulatory potential of all four JAKi currently approved for RA treatment in Europe. Therefore, we treated freshly and preactivated PBMCs with rising concentrations of either tofacitinib, baricitinib, upadacitinib, filgotinib, and MTX and determined the effect on cell proliferation, activation (CD25) and apoptosis. Furthermore, we investigated the impact of different JAKi and MTX on DNA damage induction and repair by fluorescence microscopic analysis of DNA DSB markers γH2AX and 53BP1. Our study indicates a broad comparability of

the immunomodulatory effects induced by different JAKi and offers a first indication, that (pan)JAKi may impair DNA damage repair in radiated lymphocytes.

#### **2. Experimental Section**

#### *2.1. Ethics Statement*

The study was performed in accordance with the Declaration of Helsinki and was approved by the local ethics committee (No. 183/20). All 14 healthy blood donors (10 female and 4 male; mean age: 35 ± 12 years) who agreed to participate in this study provided written informed consent.
