Several types of pathogenic bacteria produce genotoxins that induce DNA damage in host cells. Accumulating evidence suggests that a central function of these genotoxins is to dysregulate the host's immune response, but the underlying mechanisms remain unclear. To address this issue, we investigated the effects of the most widely expressed bacterial genotoxin, the cytolethal distending toxin (CDT), on T cells-the key mediators of adaptive immunity. We show that CDT induces premature senescence in activated CD4 T cells in vitro and provide evidence suggesting that infection with genotoxin-producing bacteria promotes T cell senescence in vivo. Moreover, we demonstrate that genotoxin-induced senescent CD4 T cells assume a senescence-associated secretory phenotype (SASP) which, at least partly, is orchestrated by the ATM-p38 signaling axis. These findings provide insight into the immunomodulatory properties of bacterial genotoxins and uncover a putative link between bacterial infections and T cell senescence.

Patients with Sézary syndrome (SS), a leukemic variant of cutaneous T-cell lymphoma (CTCL), are prone to Staphylococcus aureus infections and have a poor prognosis due to treatment resistance. Here, we report that S aureus and staphylococcal enterotoxins (SE) induce drug resistance in malignant T cells against therapeutics commonly used in CTCL. Supernatant from patient-derived, SE-producing S aureus and recombinant SE significantly inhibit cell death induced by histone deacetylase (HDAC) inhibitor romidepsin in primary malignant T cells from patients with SS. Bacterial killing by engineered, bacteriophage-derived, S aureus–specific endolysin (XZ.700) abrogates the effect of S aureus supernatant. Similarly, mutations in major histocompatibility complex (MHC) class II binding sites of SE type A (SEA) and anti-SEA antibody block induction of resistance. Importantly, SE also triggers resistance to other HDAC inhibitors (vorinostat and resminostat) and chemotherapeutic drugs (doxorubicin and etoposide). Multimodal single-cell sequencing indicates T-cell receptor (TCR), NF-κB, and JAK/STAT signaling pathways (previously associated with drug resistance) as putative mediators of SE-induced drug resistance. In support, inhibition of TCR-signaling and Protein kinase C (upstream of NF-κB) counteracts SE-induced rescue from drug-induced cell death. Inversely, SE cannot rescue from cell death induced by the proteasome/NF-κB inhibitor bortezomib. Inhibition of JAK/STAT only blocks rescue in patients whose malignant T-cell survival is dependent on SE-induced cytokines, suggesting 2 distinct ways SE can induce drug resistance. In conclusion, we show that S aureus enterotoxins induce drug resistance in primary malignant T cells. These findings suggest that S aureus enterotoxins cause clinical treatment resistance in patients with SS, and antibacterial measures may improve the outcome of cancer-directed therapy in patients harboring S aureus.

Staphylococcus aureus is suspected to fuel disease activity in cutaneous T-cell lymphomas. In this study, we investigate the effect of a recombinant, antibacterial protein, endolysin (XZ.700), on S. aureus skin colonization and malignant T-cell activation. We show that endolysin strongly inhibits the proliferation of S. aureus isolated from cutaneous T-cell lymphoma skin and significantly decreases S. aureus bacterial cell counts in a dose-dependent manner. Likewise, ex vivo colonization of both healthy and lesional skin by S. aureus is profoundly inhibited by endolysin. Moreover, endolysin inhibits the patient-derived S. aureus induction of IFNγ and the IFNγ-inducible chemokine CXCL10 in healthy skin. Whereas patient-derived S. aureus stimulates activation and proliferation of malignant T cells in vitro through an indirect mechanism involving nonmalignant T cells, endolysin strongly inhibits the effects of S. aureus on activation (reduced CD25 and signal transducer and activator of transcription 5 phosphorylation) and proliferation (reduced Ki-67) of malignant T cells and cell lines in the presence of nonmalignant T cells. Taken together, we provide evidence that endolysin XZ.700 inhibits skin colonization, chemokine expression, and proliferation of pathogenic S. aureus and blocks their potential tumor-promoting effects on malignant T cells.

Conventionally, the size, shape, and biomechanics of cartilages are determined by their voluminous extracellular matrix. By contrast, we found that multiple murine cartilages consist of lipid-filled cells called lipochondrocytes. Despite resembling adipocytes, lipochondrocytes were molecularly distinct and produced lipids exclusively through de novo lipogenesis. Consequently, lipochondrocytes grew uniform lipid droplets that resisted systemic lipid surges and did not enlarge upon obesity. Lipochondrocytes also lacked lipid mobilization factors, which enabled exceptional vacuole stability and protected cartilage from shrinking upon starvation. Lipid droplets modulated lipocartilage biomechanics by decreasing the tissue’s stiffness, strength, and resilience. Lipochondrocytes were found in multiple mammals, including humans, but not in nonmammalian tetrapods. Thus, analogous to bubble wrap, superstable lipid vacuoles confer skeletal tissue with cartilage-like properties without “packing foam–like” extracellular matrix.

Perturbation in JAK-STAT signaling has been reported in the pathogenesis of cutaneous T cell lymphoma (CTCL). JAK3 is predominantly associated with the intra-cytoplasmic part of IL-2Rγc located in the plasma membrane of hematopoietic cells. Here we demonstrate that JAK3 is also ectopically expressed in the nucleus of malignant T cells. We detected nuclear JAK3 in various CTCL cell lines and primary malignant T cells from patients with Sézary syndrome, a leukemic variant of CTCL. Nuclear localization of JAK3 was independent of its kinase activity whereas STAT3 had a modest effect on nuclear JAK3 expression. Moreover, JAK3 nuclear localization was only weakly affected by blockage of nuclear export. An inhibitor of the nuclear export protein CRM1, Leptomycin B, induced an increased expression of SOCS3 in the nucleus, but only a weak increase in nuclear JAK3. Importantly, immunoprecipitation experiments indicated that JAK3 interacts with the nuclear protein POLR2A, the catalytic subunit of RNA Polymerase II. Kinase assays showed tyrosine phosphorylation of recombinant human Histone H3 by JAK3 in vitro—an effect which was blocked by the JAK inhibitor (Tofacitinib citrate). In conclusion, we provide the first evidence of nuclear localization of JAK3 in malignant T cells. Our findings suggest that JAK3 may have a cytokine-receptor independent function in the nucleus of malignant T cells, and thus a novel non-canonical role in CTCL.

Keratin 8/18, the predominant keratin pair of simple epithelia, is often aberrantly expressed in various squamous cell carcinomas (SCCs) including skin SCC. Its aberrant expression is correlated with increased invasiveness and poor prognosis of the same, although the underlying mechanism is still unclear. A previous report from our laboratory has shown K8‐mediated regulation of α6β4 integrin signaling and thereby tumorigenic potential of oral SCC‐derived cells. Another study on transgenic mouse model has shown that during skin carcinogenesis, K8 favors conversion of papillomas toward malignancy. In order to understand the role of K8 and allied mechanism in skin SCC, K8 was stably knocked down in a skin epidermoid carcinoma‐derived A431 cells. K8 downregulation significantly reduced the tumorigenic potential of these cells. In agreement with our phenotypic data, differential quantitative proteomics followed by IPA analysis showed altered expression of many proteins associated with biological functions including ‘Cancer’, ‘Cellular movement’, ‘Cell death and survival’, and ‘Cellular morphology’. Some of these proteins were TMS1, MARCKSL1, RanBP1, 14‐3‐3γ, Rho‐GDI2, etc. Furthermore, to our surprise, there was a significant reduction in K17 protein stability upon loss of K8, probably due to its caspase‐mediated degradation. This was supported by altered TMS1‐NF‐κB signaling, leading to increased apoptotic sensitivity of A431 cells which in turn affected ‘Cell death and survival’. Moreover, MARCKSL1‐Paxillin1‐Rac axis was found to be deregulated bestowing a possible mechanism behind altered ‘Cellular movement’ pathway. Altogether our study unravels a much broader regulatory role of K8, governing multiple signaling pathways and consequently regulating oncogenic potential of skin SCC‐derived cells.Database: Proteome Xchange Consortium via PRIDE database (dataset identifier PXD007206).