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Veillonella parvula, prototypical member of the oral and gut microbiota, is at times commensal yet also potentially pathogenic. The definition of the molecular basis tailoring this contrasting behavior is key for broadening our understanding of the microbiota-driven pathogenic and/or tolerogenic mechanisms that take place within our body. In this study, we focused on the chemistry of the main constituent of the outer membrane of V. parvula, the lipopolysaccharide (LPS). LPS molecules indeed elicit pro-inflammatory and immunomodulatory responses depending on their chemical structures. Herein we report the structural elucidation of the LPS from two strains of V. parvula and show important and unprecedented differences in both the lipid and carbohydrate moieties, including the identification of a novel galactofuranose and mannitol-containing O-antigen repeating unit for one of the two strains. Furthermore, by harnessing computational studies, in vitro human cell models, as well as lectin binding solid-phase assays, we discovered that the two chemically diverse LPS immunologically behave differently and have attempted to identify the molecular determinant(s) governing this phenomenon. Whereas pro-inflammatory potential has been evidenced for the lipid A moiety, by contrast a plausible "immune modulating" action has been proposed for the peculiar O-antigen portion.

Dendritic cells (DCs) play a pivotal role in immune surveillance, acting as sentinels that coordinate immune responses within tissues. Although differences in the identity and functional states of DC subpopulations have been identified through multiparametric flow cytometry and single-cell RNA sequencing, these methods do not provide information about the spatial context in which the cells are located. This knowledge is crucial for understanding tissue organisation and cellular cross-talk. Recent developments in multiplex imaging techniques can now offer insights into this complex spatial and functional landscape. This review provides a concise overview of these imaging methodologies, emphasising their application in identifying DCs to delineate their tissue-specific functions and aiding newcomers in navigating this field.

Psoriasis is a chronic inflammatory skin disease with an autoimmune component and associated with joint inflammation in up to 30% of cases. To investigate autoreactive T cells, we developed an imiquimod-induced psoriasis-like inflammation model in K5-mOVA.tg C57BL/6 mice expressing ovalbumin (OVA) on the keratinocyte membrane, adoptively transferred with OT-I OVA-specific CD8+ T cells. We evaluated the expansion of OT-I CD8+ T cells and their localization in skin, blood, and spleen. scRNA-seq and TCR sequencing data from patients with psoriatic arthritis were also analyzed. In the imiquimod-treated K5-mOVA.tg mouse model, OT-I T cells were markedly expanded in the skin and blood at early time points. OT-I T cells in the skin showed mainly CXCR3+ effector memory phenotype, whereas in peripheral blood there was an expansion of CCR4+CXCR3+ OT-I cells. At a later time point, expanded OVA-specific T-cell population was found in the spleen. In patients with psoriatic arthritis, scRNA-seq and TCR sequencing data showed clonal expansion of CCR4+ TCM cells in the circulation and further expansion in the synovial fluid. Importantly, there was a clonotype overlap between CCR4+ TCM in the peripheral blood and CD8+ T-cell effectors in the synovial fluid. This mechanism could play a role in the generation and spreading of autoreactive T cells to the synovioentheseal tissues in psoriasis patients at risk of developing psoriatic arthritis.

It is well known that regulatory T-cells (Tregs) are required to prevent autoimmunity, but they may also have some less-well understood immune-stimulatory effects. In particular, in CD8(+) T-cell responses Tregs select high-affinity clones upon priming and promote memory by inhibiting inflammation-dependent generation of short-lived effector cells. In the current issue of the European Journal of Immunology [Eur. J. Immunol. 2023. 53: XXXX-XXXX], Madi et al. report the surprising finding that human and murine FOXP3(+) Tregs are a physiologically relevant source of IL-15, a homeostatic cytokine that promotes antigen-independent maintenance of CD8(+) memory T-cells. In mice that lack IL-15 selectively in FOXP3(+)Tregs the authors show that the composition of the CD8(+) T-cell memory pool is altered in the absence of Treg-derived IL-15, since a subset of terminally effector memory cells is drastically reduced. Otherwise Treg-derived IL-15 is dispensable for antiviral immune responses and the generation of anti-viral CD8(+) memory T-cells. These findings add to our understanding of the multifaceted role of Tregs in immune responses, and how IL-15 derived from different cellular sources maintains anti-viral T-cell memory.

Calcineurin (CN) inhibitors currently used to avoid transplant rejection block the activation of adaptive immune responses but also prevent the development of tolerance toward the graft, by directly inhibiting T cells. CN, through the transcription factors of the NFAT family, plays an important role also in the differentiation dendritic cells (DCs), the main cells responsible for the activation of T lymphocytes. Therefore, we hypothesized that the inhibition of CN only in DCs and not in T cells could be sufficient to prevent T cell responses, while allowing for the development of tolerance. Here, we show that inhibition of CN/NFAT pathway in innate myeloid cells, using a new nanoconjugate capable of selectively targeting phagocytes in vivo, protects against graft rejection and induces a longer graft acceptance compared to common CN inhibitors. We propose a new generation of nanoparticles-based selective immune suppressive agents for a better control of transplant acceptance.

Surgical excision followed by histopathological examination is the gold standard for melanoma screening. However, the color-based inspection of hematoxylin-and-eosin-stained biopsies does not provide a space-resolved quantification of the melanin content in melanocytic lesions. We propose a non-destructive photo-thermal imaging method capable of characterizing the microscopic distribution and absolute concentration of melanin pigments in excised melanoma biopsies. By exploiting the photo-thermal effect primed by melanin absorption of visible laser light we obtain label-free super-resolution far-infrared thermal images of tissue sections where melanin is spatially mapped at sub-diffraction 40-µm resolution. Based on the finite-element simulation of the full 3D heat transfer model, we are able to convert temperature maps into quantitative images of the melanin molar concentration on B16 murine melanoma biopsies, with 4·10−4 M concentration sensitivity. Being readily applicable to human melanoma biopsies in combination with hematoxylin-and-eosin staining, the proposed approach could complement traditional histopathology in the characterization of pigmented lesions ex-vivo.

During gram-negative septicemia, interactions between platelets and neutrophils initiate a detrimental feedback loop that sustains neutrophil extracellular trap (NET) induction, disseminated intravascular coagulation, and inflammation. Understanding intracellular pathways that control platelet-neutrophil interactions is essential for identifying new therapeutic targets. Here, we found that thrombin signaling induced activation of the transcription factor NFAT in platelets. Using genetic and pharmacologic approaches, as well as iNFATuation, a newly developed mouse model in which NFAT activation can be abrogated in a cell-specific manner, we demonstrated that NFAT inhibition in activated murine and human platelets enhanced their activation and aggregation, as well as their interactions with neutrophils and NET induction. During gram-negative septicemia, NFAT inhibition in platelets promoted disease severity by increasing disseminated coagulation and NETosis. NFAT inhibition also partially restored coagulation ex vivo in patients with hypoactive platelets. Our results define non-transcriptional roles for NFAT that could be harnessed to address pressing clinical needs.

Growing evidence suggests that conventional dendritic cells (cDCs) undergo aberrant maturation in COVID-19, which negatively affects T-cell activation. The presence of effector T cells in patients with mild disease and dysfunctional T cells in severely ill patients suggests that adequate T-cell responses limit disease severity. Understanding how cDCs cope with SARS-CoV-2 can help elucidate how protective immune responses are generated. Here, we report that cDC2 subtypes exhibit similar infection-induced gene signatures, with the upregulation of IFN-stimulated genes and IL-6 signaling pathways. Furthermore, comparison of cDCs between patients with severe and mild disease showed severely ill patients to exhibit profound downregulation of genes encoding molecules involved in antigen presentation, such as MHCII, TAP, and costimulatory proteins, whereas we observed the opposite for proinflammatory molecules, such as complement and coagulation factors. Thus, as disease severity increases, cDC2s exhibit enhanced inflammatory properties and lose antigen presentation capacity. Moreover, DC3s showed upregulation of anti-apoptotic genes and accumulated during infection. Direct exposure of cDC2s to the virus in vitro recapitulated the activation profile observed in vivo. Our findings suggest that SARS-CoV-2 interacts directly with cDC2s and implements an efficient immune escape mechanism that correlates with disease severity by downregulating crucial molecules required for T-cell activation.

In cancer microenvironment, aberrant glycosylation events of ECM proteins and cell surface receptors occur. We developed a protocol to generate 3D bioprinted models of colorectal cancer (CRC) crosslinking hyaluronic acid and gelatin functionalized with three signalling glycans characterized in CRC, 3′-Sialylgalactose, 6′-Sialylgalactose and 2′-Fucosylgalactose. The crosslinking, performed exploiting azide functionalized gelatin and hyaluronic acid and 4arm-PEG-dibenzocyclooctyne, resulted in biocompatible hydrogels that were 3D bioprinted with commercial CRC cells HT-29 and patient derived CRC tumoroids. The glycosylated hydrogels showed good 3D printability, biocompatibility and stability over the time. SEM and synchrotron radiation SAXS/WAXS analysis revealed the influence of glycosylation in the construct morphology, whereas MALDI-MS imaging showed that protein profiles of tumoroid cells vary with glycosylation, indicating that sialylation and fucosylation of ECM proteins induce diverse alterations to the proteome of the tumoroid and surrounding cells.

Collagen organization changes with the tissue pathological conditions, like cancer, and can be monitored through Second-Harmonic Generation imaging, a label-free method sensitive to the fibrils microstructure. As a consequence, collagen can be exploited as an early-tumor diagnosis marker. Coupling a phasorbased method with a non-supervised machine learning algorithm, our protocol is able to map pixel by pixel crucial features of the collagen fibrils and enlighten different collagen organizations. Basing on these maps, our protocol can automatically discriminate, on fixed tumor sections, tumor area from the surrounding tissue with an accuracy of ∼ 90%, opening the possibility to effectively assist histopathologists in cancer diagnosis.

To understand how a protective immune response against SARS-CoV-2 develops over time, we integrated phenotypic, transcriptional and repertoire analyses on PBMCs from mild and severe COVID-19 patients during and after infection, and compared them to healthy donors (HD). A type I IFN-response signature marked all the immune populations from severe patients during the infection. Humoral immunity was dominated by IgG production primarily against the RBD and N proteins, with neutralizing antibody titers increasing post infection and with disease severity. Memory B cells, including an atypical FCRL5+ T-BET+ memory subset, increased during the infection, especially in patients with mild disease. A significant reduction of effector memory, CD8+T cells frequency characterized patients with severe disease. Despite such impairment, we observed robust clonal expansion of CD8+T lymphocytes, while CD4+T cells were less expanded and skewed toward TCMand TH2-like phenotypes. MAIT cells were also expanded, but only in patients with mild disease. Terminally differentiated CD8+GZMB+effector cells were clonally expanded both during the infection and post-infection, while CD8+GZMK+lymphocytes were more expanded post-infection and represented bona fide memory precursor effector cells. TCR repertoire analysis revealed that only highly proliferating T cell clonotypes, which included SARS-CoV-2-specific cells, were maintained post-infection and shared between the CD8+GZMB+and GZMK+subsets. Overall, this study describes the development of immunity against SARS-CoV-2 and identifies an effector CD8+T cell population with memory precursor-like features.

The ability of dendritic cells (DCs) to sense viral pathogens and orchestrate a proper immune response makes them one of the key players in antiviral immunity. Different DC subsets have complementing functions during viral infections, some specialize in antigen presentation and cross-presentation and others in the production of cytokines with antiviral activity, such as type I interferons. In this review, we summarize the latest updates concerning the role of DCs in viral infections, with particular focus on the complex interplay between DC subsets and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Despite being initiated by a vast array of immune receptors, DC-mediated antiviral responses often converge towards the same endpoint, that is the production of proinflammatory cytokines and the activation of an adaptive immune response. Nonetheless, the inherent migratory properties of DCs make them a double-edged sword and often viral recognition by DCs results in further viral dissemination. Here we illustrate these various aspects of the antiviral functions of DCs and also provide a brief overview of novel antiviral vaccination strategies based on DCs targeting.

The chemokine receptor CCR4 has emerged as a skin-homing molecule important for the migration of T cells from the blood to the dermis. From our previous data on psoriasis patients, CCR4+ memory T cells emerged as a putative recirculating population between skin and blood. Here we focused our attention on the expression of CCR4 and skin-tropic molecules in the different stages of memory T cell differentiation. We analyzed the chemokine receptor profile in CD8+ and CD4+ CD45RA−CCR7+ (TCM) and CD45RA−CCR7− (TEM) cells. Subpopulations were further divided on the basis of CD62L expression, and the distribution among the subsets of the skin-homing molecule CLA (Cutaneous Lymphocyte Antigen) was evaluated. The characterization was performed on peripheral blood mononuclear cells isolated from 21 healthy subjects and 24 psoriasis patients. The results indicate that (i) the skin-homing CCR4 marker is mainly expressed in TCM cells, (ii) CCR4+ TCM cells also express high level of CLA and that (iii) the more differentiated phenotype TEM expresses CXCR3 and CCR5 but lower level of CCR4 and CLA. This indicates that progressive stages of memory T cell differentiation have profoundly different chemokine receptor patterns, with CD8+ TCM displaying a marked skin-tropic phenotype CLA+CCR4+. Differential skin-tropic phenotype between TCM and TEM cells was observed in both healthy subjects and psoriasis patients. However, patients showed an expanded circulating population of CD8+ TCM cells with phenotype CCR4+CXCR3+ that could play a role in the pathophysiology of psoriasis and possibly in disease recurrence.

Humans are constantly exposed to fungi, either in the form of commensals at epithelial barriers or as inhaled spores. Innate immune cells play a pivotal role in maintaining commensal relationships and preventing skin, mucosal, or systemic fungal infections due to the expression of pattern recognition receptors that recognize fungal cell wall components and modulate both their activation status and the ensuing adaptive immune response. Commensal fungi also play a critical role in the modulation of homeostasis and disease susceptibility at epithelial barriers. This review will outline cellular and molecular mechanisms of anti-fungal innate immunity focusing on C-type lectin receptors and their relevance in the context of host-fungi interactions at skin and mucosal surfaces in murine experimental models as well as patients susceptible to fungal infections.

Purpose: The partial ineffectiveness and side effects of inflammatory bowel disease (IBD) current therapies drive basic research to look for new therapeutic target in order to develop new drug lead. Considering the pivotal role played by toll-like receptors (TLRs) in gut inflammation, we evaluate here the therapeutic effect of the synthetic glycolipid TLR4 antagonist FP7. Methods: The anti-inflammatory effect of FP7, active as TLR4 antagonist, was evaluated on peripheral blood mononuclear cells (PBMCs) and lamina propria mononuclear cells (LPMCs) isolated from IBD patients, and in a mouse model of ulcerative colitis. Results: FP7 strongly reduced the inflammatory responses induced by lipopolysaccharide (LPS) in vitro, due to its capacity to compete with LPS for the binding of TLR4/MD-2 receptor complex thus inhibiting both the MyD88- and TRIF-dependent inflammatory pathways. Colitic mice treated with FP7 exhibit reduced colonic inflammation and decreased levels of pro-inflammatory cytokines. Conclusions: This study suggests that TLR4 chemical modulation can be an effective therapeutic approach to IBD. The selectivity of FP7 on TLR4 makes this molecule a promising drug lead for new small molecules-based treatments.

The aim of this study is to identify subsets of T cells differentially represented in the circulation of patients with psoriatic arthritis and to evaluate the possibility that they can recirculate between peripheral blood and the inflamed joints. We analyzed the phenotype and cytokine expression in circulating CD8+ and CD4+ T cells in 69 subjects: 28 with cutaneous psoriasis, 15 patients with psoriatic arthritis, and 26 healthy subjects. In the circulation, the percentage of each subset was compared among the groups and correlation was calculated with the serum concentration of C-reactive protein. To investigate the migration of T cells towards the inflamed joints, we performed a transwell migration assay towards patient serum and synovial fluid. In selected patients we analyzed in parallel T cells from peripheral blood and from synovial fluid. In the circulation, we found increased percentage of CD8+ CCR6+ T cell effectors expressing CD69 and of IL-17-producing T cells in patients with psoriatic arthritis. CD8+ effector/effector memory T cells showed increased migration towards synovial fluid. Finally, in synovial fluid we found accumulation of CXCR3+ CD8+ T cells and CD69+ cells. CD4+ T cells in the two compartments shared many similarities with CD8+ T cells. The results indicate a role for memory T cell effectors in systemic and joint manifestations of psoriatic arthritis.

TLRs are a class of pattern recognition receptors (PRRs) that detect invading microbes by recognizing pathogen-associated molecular patterns (PAMPs). Upon PAMP engagement, TLRs activate a signaling cascade that leads to the production of inflammatory mediators. The localization of TLRs, either on the plasma membrane or in the endolysosomal compartment, has been considered to be a fundamental aspect to determine to which ligands the receptors bind, and which transduction pathways are induced. However, new observations have challenged this view by identifying complex trafficking events that occur upon TLR-ligand binding. These findings have highlighted the central role that endocytosis and receptor trafficking play in the regulation of the innate immune response. Here, we review the TLR4 and TLR9 transduction pathways and the importance of their different subcellular localization during the inflammatory response. Finally, we discuss the implications of TLR9 subcellular localization in autoimmunity.

The current treatments for chronic inflammatory diseases cause severe side effects due to nonspecific drug accumulation. Nanotechnology opens the way to new therapeutic strategies that exploit the ability of immune cells, and especially of phagocytes, to internalize nanoparticles. The cellular uptake of nanoparticles requires specific interactions and is affected by the chemical and physical properties of the carriers. Therefore, optimizing these properties is crucial for designing nanodrugs for immunotherapy. In perspective, we discuss the nanoparticle-based approaches that have been proposed to induce tolerance in autoimmune disorders and lessen the symptoms of inflammatory diseases.

Viral infections of the lower respiratory tract are a leading cause of mortality. Mounting evidence indicates that most severe cases are characterized by aberrant immune responses and do not depend on viral burden. In this study, we assessed how type III interferons (IFN-l) contribute to the pathogenesis induced by RNA viruses. We report that IFN-l is present in the lower, but not upper, airways of patients with coronavirus disease 2019 (COVID-19). In mice, we demonstrate that IFN-l produced by lung dendritic cells in response to a synthetic viral RNA induces barrier damage, causing susceptibility to lethal bacterial superinfections. These findings provide a strong rationale for rethinking the pathophysiological role of IFN-l and its possible use in clinical practice against endemic viruses, such as influenza virus as well as the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Two‐photon laser writing is used here to fabricate 3D proteinaceous microstructures with photothermal functionality in the near‐infrared spectral region and tunable elasticity. The photo‐cross‐linking is initiated in bovine serum albumin (BSA) by rose bengal or methylene blue and the photo‐thermal effect arises from gold non‐spherically symmetric nanoparticles dispersed in the ink. Massive energy transfer of the plasmonic resonances of the gold nanoparticles to methylene blue prevents effective photo‐crosslinking of BSA. However, stable microstructures with photo‐thermal functionality can be fabricated in the rose bengal proteinaceous inks. On these microstructures, with a gold atom concentration as low as 1% w/w, a highly localized temperature increase can be quickly (≅1 s) reached and maintained under continuous wave laser irradiation at 800 nm. The photothermal efficiency under continuous wave laser irradiation depends on the thickness of the microstructure and can reach 12.2 ± 0.4 °C W−1 These proteinaceous microstructures represent therefore a promising platform for future applications in the fields like physical stimulation of cells for regenerative nanomedicine.

Chronic inflammation is one of the causes of neurodegeneration in Amyotrophic lateral sclerosis (ALS). Here we examined whether circulating dendritic cells (DCs) can contribute to disease progression. We found ALS patients show a significant reduction in the number of circulating DCs. Also, patients’ DCs present an increased expression of CD62L and a tendency to overexpress CCR2 compared with healthy donors. Moreover, DCs derived from a subpopulation of ALS patients produced higher levels of IL-8 and CCL-2 upon lipopolysaccharide (LPS)-stimulation. Finally, we found a significant inverse correlation between the time from onset of the pathology to its diagnosis and the levels of IL-6 secretion induced by LPS. Our data support the hypothesis, in a subpopulation of patients, DCs recruited at the diseased tissue produce high levels of CCL-2 and IL-8 and contribute to the inflammatory process promoting the recruitment of other inflammatory cells. An increased efficiency of IL-6 production may accelerate only the initial phases of disease progression. Blood DC analysis can be used to identify ALS patients with an altered course of inflammatory cell recruitment at the diseased central nervous system (CNS). The high levels of CD62L expression suggests this molecule could be a target for treatment of CNS inflammation.

Blood to skin recirculation could play a role in the pathogenesis of psoriasis. To investigate this possibility we dissected the phenotype of circulating T cells in psoriasis patients, calculated the correlation the clinical parameters of the disease and performed a parallel bioinformatics analysis of gene expression data in psoriatic skin. We found that circulating CCR6+ CD4+ TEM and TEFF cells significantly correlated with systemic inflammation. Conversely, the percentage of CXCR3+ CD4+ TEM cells negatively correlated with the severity of the cutaneous disease. Importantly CLA+ CD4+ TCM cells expressing CCR6+ or CCR4+ CXCR3+ negatively correlated with psoriasis severity suggesting recruitment to the skin compartment. This assumption was reinforced by gene expression data showing marked increase of CCR7 and CLA-encoding gene SELPLG expression in psoriatic skin and strong association of their expression. The data enlightens a role for CD4+ T cells trafficking between blood and skin in cutaneous and systemic manifestations of psoriasis.

Nanoparticles represent a new generation of drug delivery systems that can be engineered to harness optimal target selectivity for specific cells and tissues and high drug loading capacity, allowing for improved pharmacokinetics and enhanced bioavailability of therapeutics. The spontaneous propensity of both organic and colloidal nanoparticles to be captured by the cells of the reticuloendothelial system encouraged their utilization as passive targeting systems that can be preferentially directed to innate immune cells, such as macrophages, dendritic cells and neutrophils. The natural affinity for phagocytic cells suggests the possible implementation of nanoparticles as an immunotherapeutic platform for inflammatory diseases and autoimmune disorders. Here we discuss the recent advances in the application of nanotechnology to induce antigen-specific tolerance in autoimmunity and the use of nanoparticles for anti-inflammatory therapies, including treatment of inflammatory bowel diseases, psoriasis and rheumatoid arthritis.

Vaccines represent the discovery of utmost importance for global health, due to both prophylactic action to prevent infections and therapeutic intervention in neoplastic diseases. Despite this, current vaccination strategies need to be refined to successfully generate robust protective antigen-specific memory immune responses. To address this issue, one possibility is to exploit the high efficiency of dendritic cells (DCs) as antigen-presenting cells for T cell priming. DCs functional plasticity allows shaping the outcome of immune responses to achieve the required type of immunity. Therefore, the choice of adjuvants to guide and sustain DCs maturation, the design of multifaceted vehicles, and the choice of surface molecules to specifically target DCs represent the key issues currently explored in both preclinical and clinical settings. Here, we review advances in DCs-based vaccination approaches, which exploit direct in vivo DCs targeting and activation options. We also discuss the recent findings for efficient antitumor DCs-based vaccinations and combination strategies to reduce the immune tolerance promoted by the tumor microenvironment.

Toll-like receptor 4 (TLR4) activation is pivotal to innate immunity and has been shown to regulate proliferation and differentiation of human neural stem cells (hNSCs) in vivo. Here we study the role of TLR4 in regulating hNSC derived from the human telencephalic-diencephalic area of the fetal brain and cultured in vitro as neurospheres in compliance with Good Manifacture Procedures (GMP) guidelines. Similar batches have been used in recent clinical trials in ALS patients. We found that TLR2 and 4 are expressed in hNSCs as well as CD14 and MD-2 co-receptors, and TLR4 expression is downregulated upon differentiation. Activation of TLR4 signaling by lipopolysaccharide (LPS) has a positive effect on proliferation and/or survival while the inverse is observed with TLR4 inhibition by a synthetic antagonist. TLR4 activation promotes neuronal and oligodendrocyte differentiation and/or survival while TLR4 inhibition leads to increased apoptosis. Consistently, endogenous expression of TLR4 is retained by hNSC surviving after transplantation in ALS rats or immunocompromised mice, thus irrespectively of the neuroinflammatory environment. The characterization of downstream signaling of TLR4 in hNSCs has suggested some activation of the inflammasome pathway. This study suggests TLR4 signaling as essential for hNSC self-renewal and as a novel target for the study of neurogenetic mechanisms

After LPS recognition, the MyD88-dependent and the TRIF-dependent pathways are consecutively activated in macrophages. Schappe et al. (2018) show that the chanzyme TRPM7 is required for an efficient LPS receptor complex endosomal relocation and the activation of the TRIF pathway.

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.