Cutaneous T-cell lymphoma (CTCL) represents a heterogeneous group of non-Hodgkin lymphomas characterised by the infiltration and expansion of neoplastic mature T cells, primarily in the skin (1). In these lymphoproliferative disorders, an impaired immune system is primarily responsible for more recurrent infections, chronic inflammation, and the suppression of antitumor activity (2, 3). External factors have been proposed as one of the key reasons for the aggravation of the disease (3–5). A recent hypothesis proposes that microbial antigens play a role in promoting chronic inflammation and malignant cell transformation (6); a similar role has been described in several other skin diseases, such as atopic dermatitis, psoriasis, and acne vulgaris (7–10). Advances in modern technologies, and especially in the sequencing methods such as 16s sequencing and whole-genome shotgun sequencing (11), equip researchers with more tools they can use to understand changes in skin-microbial populations up to the species level in different CTCL disease stages and during therapeutic interventions.

Intact human skin provides an effective barrier against environmental effects. The superficial layer of human skin is colonised by a plethora of microorganisms, comprising bacteria, archaea, fungi, and viruses, which form a mutualistic symbiosis. The balance of this heterogenous microbial community is essential for protecting the organism against invading pathogens and the breakdown of natural products (12). The microbiota can modulate the production of various anti-microbial peptides (AMPs), cytokines, and chemokines in the skin (5, 9, 12, 13). Commensal skin microbiota, including bacteria such as Staphylococcus hominis (SH) or S. epidermidis (SE) (14), ubiquitously colonise human skin and are non-harmful to humans. In contrast, Staphylococcus aureus (SA) is associated with various pathogenic skin conditions in humans. SA-derived enterotoxins have evoked particular interest because they belong to a class of “superantigens”, which are exceptionally potent activators of T cells. If the skin barrier is breached or the immune system is impaired due to the expansion of malignant T cells in the skin (as in the case of CTCL), this delicate balance between commensal and pathogenic microorganisms is disrupted; this, in turn, triggers chronic inflammation which aggravates the disease phenotype. Furthermore, a chronic pro-inflammatory micro-environment has been shown to promote malignant T cell proliferation (2, 6, 15).

Most patients with early-stage mycosis fungoides (MF), the most common form of CTCL, characteristically present with cutaneous patches and plaques. These patients are treated with phototherapy to clear skin lesions and increase their disease-free survival rates. Narrowband UVB (NB-UVB) and psoralen plus UVA (PUVA) are the two primary forms of phototherapy used to treat these CTCL patients (16–21). Regarding leukemic CTCL (L-CTCL), extracorporeal photopheresis (ECP) is usually prescribed (21, 22) as therapy. UV radiation has been experimentally proven to have profound qualitative and quantitative influences on the composition of the skin microbiome (23, 24). Moreover, a study from our lab showed that the skin microbiome regulates the effect of UV radiation on cellular response and immune function (25). For these reasons, understanding the skin microbiome and host-microbial interactions during the phototherapeutic treatment regime in CTCL is extremely important. For instance, we have recently shown that PUVA induces local type 1 interferon production and antitumor response in CTCL patients, and intriguingly, rescued deficient interferon production may help in fighting infection with Staphylococcus aureus (SA), a driving factor in the pathophysiology of the disease (26).

The dysbiosis observed in the microbiome of CTCL patients is considered more than mere coincidence. Studies suggest that alterations in the microbiome can influence immune dysregulation, inflammation, and the progression of CTCL (4, 9, 15). Furthermore, microbial metabolites have been found to modulate T-cell responses and affect tumour microenvironments, potentially impacting disease outcomes (27, 28). The cause of malignant cell transformation in CTCL remains to be elucidated, but multiple factors are associated with the disease progression, such as chromosomal aberrations, oncogenic mutations, environmental factors, and the microbiome. Increased STAT3/5 signalling, resulting from copy number gains on chromosome 17q, and the loss of negative regulators along the JAK/STAT pathway, such as suppressors of STAT1 and SOCS1, are possibly key genetic factors that contribute to increased clonal expansion in leukemic cutaneous T-cell lymphoma. Due to these genetic abnormalities in the T cell, the immune defence system becomes severely impaired, and patients with advanced forms of the disease often die because of infection rather than lymphoma. Previous studies have demonstrated the crucial role of the microbiome in modulating disease activity in CTCL. Notably, Fanok et al. (29) showed in a lymphoma mouse model comparing germ-free vs. SPF animals that the presence of microbiota can significantly accelerate disease progression.

Their findings indicate that CTCL development is markedly slower under sterile conditions, highlighting the detrimental impact of bacterial presence on disease severity. This pivotal study underscores the interaction between the microbiome and immune response in CTCL, aligning with our current investigation. Our research, employing a distinct model and interventions, further explores this link, providing additional evidence that modulating the skin microbiome through antibiotics and phototherapy can delay tumor growth and improve survival outcomes. These independent yet complementary approaches reinforce the concept that targeting microbial influences offers a promising avenue for therapeutic intervention in CTCL (29). Intriguingly, severe bacterial infections are often seen in CTCL patients because malignant T cells also may induce significant changes in the skin architecture; this, in turn, impairs the skin barrier function, increasing the patient’s susceptibility to bacterial infections and their spread. Recent research, has provided insight into this process, showing how primary malignant T cells induce significant changes in the expression of skin barrier proteins in CTCL through cytokine-mediated JAK/STAT signaling, highlighting the intricate relationship between malignant T cell activity and compromised skin barrier integrity in the disease pathology of CTCL (30). SA has also been presumed to play a tumor-promoting role since antibiotic treatment specific to SA has been shown to have an inhibitory effect on the tumor burden in some patients. This observation aligns with the findings by Gluud et al. (31), further supporting the notion of targeted antibiotic therapy as a viable approach to modulate disease progression in CTCL (32).

This study was carried out to elucidate the host-microbial interaction occurring during the phototherapeutic treatment of CTCL, to support the rationale for modulating the microbial milieu during the disease course of CTCL, and to indicate the therapeutic potential of such modulation.

New evidence suggests that the skin microbiome in CTCL patients differs significantly from that of healthy individuals. Studies have reported decreased microbial diversity and alterations in the relative abundance of specific bacterial species in CTCL-affected skin (5, 9, 33). Notably, Staphylococcus aureus (SA) colonisation (6, 35) was observed in a subset of CTCL patients, exacerbating inflammation and contributing to disease progression. In CTCL, pronounced erythema in the lesional skin was associated with an increase in SA colonisation (6, 15, 35), and severe pain and lesion thickness were associated with the presence of Corynebacterium sp. and Pelomonas sp (33).

By using the EL4 T-cell lymphoma cutaneous mouse transplantation model, we could elucidate host-microbial interactions in CTCL during a phototherapeutic treatment regime and study the modulation of these interactions through antibiotic treatment. We observed that triple antibiotic treatment significantly delayed tumour occurrence and growth, which prolonged the survival of mice in the model, irrespective of the allocation to standard therapeutic agents (PUVA, UVB). An analysis of the beta diversity index obtained by applying the Bray−Curtis model showed that the microbial population significantly changed upon antibiotic treatment. This change was linked to an increase in the numbers of T_reg-inducing commensal Clostridium species (12, 34) and a significant reduction in the numbers of the facultative pathogenic Corynebacterium, Pelomonas, Streptococcus, Pseudomonas, and Cutibacterium species. Interestingly, we observed a significant decrease in the detection frequency of Staphylococcus aureus but an increase in overall number of Staphylococcus genus, indicating that antibiotic treatment helped mice to regain microbial balance and increased the numbers of non-pathogenic Staphylococcus populations, which per se enables the mice to regain microbial eubiosis state (27).

Given the observed dysbiosis and the potential role of the microbiome in CTCL progression, the use of antibiotics may be proposed as a therapeutic approach. Antibiotics could modulate the microbiome, target specific bacterial species, or alter the microbial milieu to favour a more beneficial composition. The complex interplay between the immune system and the microbiome has been recognised by an increasing number of investigators as having a significant impact on human health and disease (35). In recent years, researchers have begun to shed light on the role of the microbiome in CTCL and the potential use of antibiotic intervention to modulate the microbiome in managing this disease (5, 31).

We have explored the impact of the skin microbiome modulation, through antibiotics and phototherapy, on disease progression in CTCL. In light of the groundbreaking findings by Vadivel et al. (36), demonstrating that S. aureus can induce drug resistance in malignant T cells, our study gains additional significance. This resistance is mediated through pathways previously implicated in CTCL pathogenesis and treatment resistance, notably TCR, NFκB, and JAK/STAT signaling. Given the established antibacterial effects of phototherapy, its role in CTCL treatment may extend beyond direct antineoplastic activity, potentially also mitigating bacterial-driven drug resistance. Vadivel et al.’s findings underscore the complex interplay between microbial pathogens, immune responses, and therapeutic resistance in CTCL. Therefore, our data suggest that combining phototherapy with targeted antimicrobial strategies could offer a twofold benefit: direct tumor cell cytotoxicity and disruption of bacterial-mediated resistance mechanisms. This approach, opens new avenues for enhancing the efficacy of existing and future therapeutic strategies against CTCL, warranting further investigation (36).

In another recent pivotal study by Liu et al. (37), the characteristics of S. aureus colonization in CTCL were extensively examined, revealing significant findings that enhance our current understanding of the microbiome’s impact on CTCL. The study included a substantial cohort of over 60 patients, providing a robust dataset for analysis. Liu et al. discovered that S. aureus colonization was present in a significant portion of CTCL patients and that the colonization rates increased with disease progression. Notably, the study also compared lesional and contralateral non-lesional skin sites, finding that S. aureus colonization was more prevalent in lesional skin, which may suggest a potential role of bacterial presence in exacerbating disease severity (37). These findings align with our current study’s hypothesis on the microbiome’s influence on CTCL progression and highlight the importance of considering microbial factors in developing therapeutic strategies. Such insights are invaluable for informing future research and clinical practices, suggesting a potential benefit in targeting microbial colonization as part of comprehensive CTCL treatment plans.

The recent advancements in understanding the interplay between microbial interactions and immune responses have significantly enriched our perspective on CTCL management. The recent study in collaboration with our lab by Yu et al. (26) and the commentary by Goel and Rook (38), have illuminated the promising role of PUVA therapy in enhancing type I IFN responses, a pathway also implicated in the antimicrobial response against Staphylococcus aureus as demonstrated by research highlighting the bacterium’s activation of type I IFN signaling through both its Xr domain (39) and via TLR9 in dendritic cells (40). Furthermore, the study on gamma interferon’s role in bolstering human endothelial cells against S. aureus infection (41) underscores the critical nature of IFN signaling in mediating resistance to microbial infections. These insights not only underscore the therapeutic potential of targeting microbial interactions and immune pathways in CTCL but also suggest a broader applicability in enhancing antitumor immunity and patient outcomes through integrated approaches that consider the microbiome’s influence on disease progression and treatment response.

In a human study, the bacterial groups Bacteroides, Escherichia/Shigella, and Streptococcus were found to be most prevalent in disease conditions such as polycystic ovary syndrome (PCOS) and obesity (42). In contrast, Akkermansia and Ruminococcaceae decreased in PCOS and showed opposite results for body weight, sex hormones, and brain-intestinal peptides (42, 43). In our study, also we see a significant decrease in Escherichia/Shigella and an increase in the detection frequency of Akkermansia and Ruminococcaceae upon antibiotic intervention, indicating the role of these microorganisms in disease severity in our model.

The mechanisms underlying the potential efficacy of antibiotics in CTCL are multifaceted. Antibiotics may directly affect bacterial species associated with disease progression, reduce pro-inflammatory stimuli, modulate immune responses, and alter the tumour microenvironment (15, 31). Additionally, antibiotics might influence the production of microbial metabolites that impact T-cell function and immune surveillance (31). For this reason, further host-microbial interaction studies are essential to provide support for the use of specific antibiotic treatment to mitigate CTCL symptoms. Further research should also focus on developing antibiotics or anti-microbial agents with improved specificity and a reduced impact on the commensal microbiome. Targeting specific bacteria associated with CTCL progressions, such as SA, Corynebacterium sp., and Pelomonas sp. while preserving the beneficial microbiota could provide a more precise approach.

Assessing the long-term effects of antibiotic interventions in CTCL is crucial. Prospective studies are needed to evaluate the extent of microbiome modulation, potential microbiome recovery after antibiotic cessation, and the impact of this treatment on disease progression and overall patient outcomes. The use of precision medicine strategies, including microbial profiling, genomics, and metabolomics (44), can help to identify patients who are more likely to respond to antibiotic interventions. Understanding the individualised characteristics of the microbiome and its interactions with the host immune system could also improve treatment outcomes.

While evidence suggests the clinical benefits of such treatments, further research is needed to elucidate the optimal antibiotic regimens, potential side effects, and long-term implications of antibiotic use. Understanding the complex interplay between the microbiome, antibiotics, and CTCL will contribute to the development of personalised treatment strategies for this challenging disease. We believe that this research describes a rationale for using specific antibiotic interventions to modulate the microbial milieu during the disease course of CTCL and indicates the therapeutic potential of such modulation. In fact, using specific antibiotics may be more effective than eradicating the entire cutaneous microbiome by using other disinfection methods, such as antiseptic whirlpool baths (27), preserve the landscape of commensals and ultimately contribute to a balanced immune response by supporting the production and release of anti-microbial peptides (24, 28, 45, 46).

One limitation of our work is that we did not perform 16s microbiome analysis of healthy untreated mice from our animal housing for baseline control purposes. However, we can refer to existing literature to understand the typical microbial diversity in the skin of normal C57BL/6 mice. Indeed, the gut and skin microbiome of C57BL/6 mice have been extensively studied, revealing that they host a diverse milieu of microorganisms. However, it is important to note that even the ‘normal microbiota’ can vary based on several factors, including the environment, diet, and genetics of the mice (47–50). For example, it was found that significant differences in microbiome of C57BL/6 mice from different vendors, indicating variability even within the same strain (47, 48). Moreover, it was demonstrated the impact of environmental factors affects the skin microbiome and immune signatures in C57BL/6 mice (49–51). Furthermore, Naik et al. highlighted the dynamic interaction between commensal microbiota and the cutaneous immune system, which is relevant to our study’s context of cutaneous T-cell lymphoma (50). Another limitation of our study is that its design did not resemble a treatment schedule once CTCL is diagnosed since it was started before tumor cell inoculation. However, the rationale for initiating antibiotic therapy 3 days prior to the tumoral challenge in our study was twofold: to establish a homogeneous microbial environment at the outset of tumor development and to assess the prophylactic and therapeutic potential of microbiome modulation in CTCL.

The skin microbiota could be modulated in several ways, including antibiotics (31), UV-C lamps (52–54), specific bacterial therapy (14, 55), probiotics (56, 57), or endolysin (58). Our study serves as an indicator that there is an unmet need for modulation of the microbiota along with conventional therapeutic approaches to reduce disease severity and improve survival.

The datasets presented in this study can be found in online repositories. Sequence data were deposited in the European Nucleotide Archive (ENA; BioProject No. PRJEB64180).

The animal study was approved by Austrian Federal Ministry of Science, Research and Economy (approval no. BMBWF-66.010/0042-V/3b/2019). The study was conducted in accordance with the local legislation and institutional requirements.

SD: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. PV-G: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Software, Visualization, Writing – review & editing. AJ: Writing – review & editing. ST: Formal analysis, Software, Writing – review & editing. PW: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing – review & editing.