Infections in the monogenic autoimmune syndrome APECED.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is caused by mutations in the Autoimmune Regulator (AIRE) gene, which impair the thymic negative selection of self-reactive T-cells and underlie the development of autoimmunity that targets multiple endocrine and non-endocrine tissues. Beyond autoimmunity, APECED features heightened susceptibility to certain specific infections, which is mediated by anti-cytokine autoantibodies and/or T-cell driven autoimmune tissue injury. These include the 'signature' APECED infection chronic mucocutaneous candidiasis (CMC), but also life-threatening coronavirus disease 2019 (COVID-19) pneumonia, bronchiectasis-associated bacterial pneumonia, and sepsis by encapsulated bacteria. Here we discuss the expanding understanding of the immunological mechanisms that contribute to infection susceptibility in this prototypic syndrome of impaired central tolerance, which provide the foundation for devising improved diagnostic and therapeutic strategies for affected patients.

MPEG1/Perforin-2 Haploinsufficiency Associated Polymicrobial Skin Infections and Considerations for Interferon-γ Therapy.

Introduction: Macrophage expressed gene 1 (MPEG1) is highly expressed in macrophages and other phagocytes. The gene encodes a bactericidal pore-forming protein, dubbed Perforin-2. Structural-, animal-, and cell-based studies have established that perforin-2 facilitates the destruction of phagocytosed microbes upon its activation within acidic phagosomes. Relative to wild-type controls, Mpeg1 knockout mice suffer significantly higher mortality rates when challenged with gram-negative or -positive pathogens. Only four variants of MPEG1 have been functionally characterized, each in association with pulmonary infections. Here we report a new MPEG1 non-sense variant in a patient with the a newly described association with persistent polymicrobial infections of the skin and soft tissue. Case Description: A young adult female patient was evaluated for recurrent abscesses and cellulitis of the breast and demonstrated a heterozygous, rare variant in MPEG1 p.Tyr430*. Multiple courses of broad-spectrum antimicrobials and surgical incision and drainage failed to resolve the infection. Functional studies revealed that the truncation variant resulted in significantly reduced capacity of the patient's phagocytes to kill intracellular bacteria. Patient-derived macrophages responded to interferon gamma (IFN-γ) by significantly increasing the expression of MPEG1. IFN-γ treatment supported perforin-2 dependent bactericidal activity and wound healing. Conclusions: This case expands the phenotype of MPEG1 deficiency to include severe skin and soft tissue infection. We showed that haploinsufficiency of perforin-2 reduced the bactericidal capacity of human phagocytes. Interferon-gamma therapy increases expression of perforin-2, which may compensate for such variants. Thus, treatment with IFN-γ could help prevent infections.

TRAF1 suppresses antifungal immunity through CXCL1-mediated neutrophil recruitment during Candida albicans intradermal infection.

BACKGROUND: Candida albicans is the most common opportunistic human fungal pathogen. The chemokine ligand CXCL1 plays a protective role in fungal infection through the recruitment of neutrophils. TRAF1 (tumor necrosis factor-associated factor 1) can be highly induced by proinflammatory stimuli such as LPS and TNF and has been implicated in septic shock. However, the role of TRAF1 in infection, especially fungal infection, remains elusive. Herein, we reveal that TRAF1 suppresses the antifungal immune response to Candida albicans intradermal infection through the regulation of CXCL1 induction and neutrophil recruitment. METHODS: A mouse model of C. albicans intradermal infection was established. The Traf1-/- mice and Traf1-/- immortalized human keratinocytes were generated. The p65 inhibitor triptolide, STAT1 inhibitor fludarabine, neutrophil-depletion antibody Ly6G, and neutralizing antibody for CXCL1 were utilized. The expression of proinflammatory cytokines and chemokines was assessed by real-time PCR and ELISA, and the activation of signaling molecules was analyzed by Western blotting. Hematoxylin and eosin staining and periodic acid Schiff staining were used for histology or fungal detection, respectively. The immunofluorescence and flow cytometry analyses were employed in the assessment of immune cell infiltration. Bone marrow transplantation and adoptive transfer experiments were conducted to establish a role for TRAF1 in the macrophage compartment in fungal skin infection. RESULTS: TRAF1-deficient mice demonstrated improved control of Candida albicans intradermal infection, and concomitant increase in neutrophil recruitment and reduction in fungal burden. The chemokine CXCL1 was upregulated in the TRAF1-deficient macrophages treated with heat-killed C. albicans. Mechanistically, TRAF1-deficient macrophages showed increased activation of transcription factor NFκB p65. The human CXCL8 was also highly induced in the TRAF1-deficient human keratinocytes upon TNF stimulation through decreasing the activation of transcription factor STAT1. TRAF1-deficient macrophages played a critical role in containing the C. albicans skin infection in vivo. CONCLUSION: TRAF1-deficient mice can better control fungal infection in the skin, a process attributable to the CXCL-neutrophil axis. Mechanistically, TRAF1 likely regulates CXCL1 expression in both macrophages and keratinocytes through the transcriptional factor NFκB and STAT1, respectively. Our finding offers new insight into the understanding of the immune regulatory mechanisms in host defense against C. albicans infection.

Infection and tissue repair of experimental cutaneous candidiasis in diabetic mice.

PURPOSE: Diabetic patients seem to be predisposed to cutaneous candidiasis. In this study, we evaluated the interference of diabetic conditions in alloxan-induced diabetic mice in relation to the development of C. albicans infection, density of M1 and M2 macrophages, distribution of collagen type I and III and anti-inflamamatory cytokines involved in tissue repair. METHODOLOGY: The mice were treated with intravenous alloxan, and all animals with blood glucose levels >250 mg dl-1 were inoculate with C. albicans intradermally in the hind paw and were studied for up to 21 days. Control groups without alloxan were used. The fungal burden was evaluated by periodic acid-Schiff (PAS) and by counting the colony forming units. Total population of macrophages were targeted with antibody to F4/80 antigen and M2 macrophages with anti-arginase antibody. Anti-inflammatory cytokines from popliteal lymph nodes were determined by capture ELISA procedures. Picrosirius red staining allowed qunantification of collagen types I and III in the infected skin by using a polarized light microscope.Results/Key findings. Diabetic mice, versus non-diabetic mice, showed a significant lower density of F4/80 and M2 macrophages, higher fungal burden, deficiency in interleukin (IL)-4 production, and delayed IL-13 responses. The later clearance of C. albicans enhanced tissue injury, leading to a decrease in collagen type I. Moreover, collagen type III was increased by interference of IL-13 and transforming growth factor-ß cytokines. CONCLUSION: These findings highlight some important changes in diabetic animal responses to C. albicans infection that may be important to the pathophysiological processes underpinning cutaneous candidiasis in diabetic patients.

In situ photoimmunotherapy for cutaneous granuloma caused by itraconazole-resistant Candida guilliermondii.

Cutaneous granulomas caused by Candida guilliermondii are difficult to cure. In situ photoimmunotherapy (ISPI) is a novel method composed of local photothermal therapy and immunoadjuvant. In this study, ISPI was used the first time clinically for cutaneous granuloma caused by itraconazole-resistant C.guilliermondii. A 10-week cycle of ISPI was composed of (1) 5% imiquimod applied topically every other day and (2) irradiation of lesions with an 808-nm diode laser at Days 14, 28, 42, and 56. Here we report our first case. A patient was treated with ISPI for four cycles. After the treatment, the lesions were eliminated without recurrence during a 12-month follow-up. Our results demonstrate that ISPI can be used as an effective treatment modality for cutaneous fungal granuloma.

Folliculocentric cutaneous presentation of disseminated Candida krusei infection in a patient with acute myeloid leukemia.

Candida krusei (C. krusei) is a multidrug-resistant opportunistic fungal pathogen that may cause disseminated infections in immunocompromised hosts. However, its clinical and histologic features are not well-characterized. We present a unique case to contribute to the growing knowledge base associated with this organism. During hospitalization for neutropenic fever, a 19-year-old man with acute myeloid leukemia, who underwent hematopoietic stem cell transplantation, developed a generalized folliculocentric eruption following initiation of antifungal therapy for newly diagnosed C. krusei fungemia. Despite adequate antifungal coverage and negative blood cultures, the follicular-based erythematous papules persisted. Biopsies demonstrated yeast within ruptured follicles, without angiotropism or involvement of the interfollicular dermis, subcutaneous tissue, or stratum corneum. Concurrent skin tissue cultures confirmed C. krusei. The patient remained febrile despite aggressive antifungal therapy, with relapse of leukemia and subsequent death. Our case is unusual given the development of cutaneous lesions following clearance of fungemia, with yeast limited to ruptured follicular lumina, possibly indicating a primary cutaneous source or early transfollicular/transepidermal elimination. Given the limited available descriptions of cutaneous histopathology for C. krusei, we seek to add to the understanding of its pathophysiology and aid in the diagnosis and treatment of this often fatal infection.

Immunological and histopathological characterization of cutaneous candidiasis.

Chronic mucocutaneous candidiasis constitutes a heterogeneous group of syndromes, characterized by non-invasive infection of the skin, nails and mucosal membranes by the fungus Candida spp. Although symptoms are heterogeneous, in all cases there is a reduction in protective cytokines, favouring the development of disease. The normal role of cytokines in skin lesions is not well understood. The present study aimed to investigate the progression of disease, understand specific cellular and molecular components involved in immunity to Candida albicans and determine the balance between pro- and anti-inflammatory cytokines over the course of cutaneous infection in immunocompetent mice. BALB/c mice (five per group) were inoculated with 5 × 10(6)C. albicans pseudohyphae in the deep dermis of the paw and analysed over 1-14  days post-infection. The contralateral paws were used for negative controls. Haematoxylin and eosin staining of skin sections during C. albicans infection was performed to analyse structural modifications to the epidermis such as hyperplasia, and infiltration of neutrophils and fibroblasts in the dermis. The cytokine populations were determined by capture ELISA using popliteal lymph node tissue. Pro-inflammatory cytokines (IL-6, TNF-α, IL-12, IFN-γ and IL-17) were detected at significant levels during the initial phase of cutaneous infection and correlated with the rapid elimination of C. albicans. Anti-inflammatory cytokines (IL-13, IL-4, IL-10 and transforming growth factor-ß) were detected on day 4 post-infection, and prevented exacerbation of inflammation and participated in healing of lesions. Thus, a balance between pro- and anti-inflammatory cytokines was fundamental for the resolution of infection. Importantly, these findings broaden our understanding of the immune mechanisms involved in chronic cutaneous candidiasis.

Signaling through IL-17C/IL-17RE is dispensable for immunity to systemic, oral and cutaneous candidiasis.

Candida albicans is a commensal fungal microbe of the human orogastrointestinal tract and skin. C. albicans causes multiple forms of disease in immunocompromised patients, including oral, vaginal, dermal and disseminated candidiasis. The cytokine IL-17 (IL-17A) and its receptor subunits, IL-17RA and IL-17RC, are required for protection to most forms of candidiasis. The importance of the IL-17R pathway has been observed not only in knockout mouse models, but also in humans with rare genetic mutations that impact generation of Th17 cells or the IL-17 signaling pathway, including Hyper-IgE Syndrome (STAT3 or TYK2 mutations) or IL17RA or ACT1 gene deficiency. The IL-17 family of cytokines is a distinct subclass of cytokines with unique structural and signaling properties. IL-17A is the best-characterized member of the IL-17 family to date, but far less is known about other IL-17-related cytokines. In this study, we sought to determine the role of a related IL-17 cytokine, IL-17C, in protection against oral, dermal and disseminated forms of C. albicans infection. IL-17C signals through a heterodimeric receptor composed of the IL-17RA and IL-17RE subunits. We observed that IL-17C mRNA was induced following oral C. albicans infection. However, mice lacking IL-17C or IL-17RE cleared C. albicans infections in the oral mucosa, skin and bloodstream at rates similar to WT littermate controls. Moreover, these mice demonstrated similar gene transcription profiles and recovery kinetics as WT animals. These findings indicate that IL-17C and IL-17RE are dispensable for immunity to the forms of candidiasis evaluated, and illustrate a surprisingly limited specificity of the IL-17 family of cytokines with respect to systemic, oral and cutaneous Candida infections.

The biological significance of TLR3 variant, L412F, in conferring susceptibility to cutaneous candidiasis, CMV and autoimmunity.

OBJECTIVE: Toll-like receptors, a major component of the innate immune system, play an important role in the initial response against pathogens. Genetic abnormalities in some receptors like TLR2, TLR3 and TLR4 have been associated with susceptibility to fungal and viral infections while other aberrations in TLR genes such as TLR3, TLR7 and TLR9 may predispose to autoimmunity. Recently we have shown an association of a TLR3 receptor variant, L412F, to susceptibility to chronic candidiasis, recurrent viral and bacterial infections and autoimmunity. We investigated here the biological implications of this TLR3 mutant. METHODS: To study the functional impact of the L412F variant of TLR3 we tested patients' peripheral blood mononuclear cells (PBMCs) as well as fibroblasts for secretion of cytokines in response to TLR3 ligand, candida or cytomegalovirus (CMV). In addition, the P2.1 cell line was used as a model for the TLR3 WT and L412F variant receptors function. RESULTS: Patient's cells carrying the L412F variant showed reduced IFNγ as well as TNFα secretion in response to stimulation with the TLR3 ligand, CMV or Candida albicans. Fibroblasts with the L412F variant showed decreased secretion of IFNλ in response to stimulation with both polyinosine ploycytidylic acid (Poly I:C) and CMV and P2.1 cells transfected with the L412F variant showed reduced secretion of IFN-ß in comparison to cells transfected with the wild type receptor. CONCLUSION: We have shown here aberrant biological responses mediated by the TLR3 variant receptor, L412F, which may explain in part susceptibility of patients to chronic candidiasis, viral infections and autoimmunity.

IL-22 and TNF-α represent a key cytokine combination for epidermal integrity during infection with Candida albicans.

T cells exercise their full impact on target cells through a combination of secreted cytokines. The recently described T helper cell subset Th22 is characterized by a combinatorial secretion of IL-22 and TNF-α. Here, we demonstrate that IL-22 increases the TNF-α-dependent induction and secretion of several immune-modulatory molecules such as initial complement factors C1r and C1s, antimicrobial peptides S100A7 and HBD-2 (human ß defensin 2), and antimicrobial chemokines CXCL-9/-10/-11 in primary human keratinocytes. The synergism of IL-22 and TNF-α is transmitted intracellularly by MAP kinases and downstream by transcription factors of the AP-1 family. The induction of innate immunity is relevant in an in vitro infection model, where keratinocytes stimulated with Th22 supernatants or recombinant IL-22 plus TNF-α effectively inhibit the growth of Candida albicans and maintain survival of epithelia. Accordingly, the combinatorial stimulation of keratinocytes with IL-22 and TNF-α most efficiently conserves the integrity of the epidermal barrier in a three-dimensional skin infection model as compared with IFN-γ, IL-17, IL-22 or TNF-α alone. In summary, we demonstrate that IL-22 and TNF-α represent a potent, synergistic cytokine combination for cutaneous immunity.

IL-23 and IL-17A, but not IL-12 and IL-22, are required for optimal skin host defense against Candida albicans.

IL-23 and Th17 cells play important roles in host defense against systemic infections with extracellular bacteria and fungi, although their roles in immunity against localized skin infections are less well defined. Here, the contributions of IL-23 and Th17 cytokines in host defense against cutaneous Candida albicans infection were evaluated. Mice deficient in IL-23 or IL-17A demonstrated delayed healing and decreased IL-17A production after skin infection with C. albicans compared with wild-type mice or mice deficient in IL-12 or IL-22. Histologic examination revealed epidermal hyperplasia overlying infected dermis four days postinoculation in wild-type mice. In IL-23-deficient mice, fungal burden was greater in skin, neither IL-17A nor IL-22 mRNAs were expressed postinfection, and these mice demonstrated only minimal epidermal hyperplasia. Exogenous recombinant IL-17A injected at the site of skin infection promoted more rapid healing of candidiasis in both wild-type mice and mice deficient in IL-23 and IL-12. Taken together, these results demonstrate that IL-23 and IL-17A, but not IL-12 and IL-22, are required for optimal host defense against cutaneous candidiasis. In addition, recombinant IL-17A may serve as a potential therapy to enhance healing in individuals with chronic cutaneous candidiasis.

Candida krusei fungemia in an immunocompromised patient.

Candida krusei is an emerging fungal pathogen found primarily in immunocompromised patients. Intrinsic resistance to fluconazole and decreasing susceptibility to other anti-fungal agents are problematic. When colonization occurs, dissemination may follow rapidly. We present a case of a patient with acute lymphoblastic leukemia who, despite being treated prophylactically with fluconazole, developed disseminated C. krusei.

Atopic dermatitis with mononuclear phagocytic activity deficiency.

Five patients with atopic dermatitis, three males and two females, aged 2 to 17 years, had positive reactions to air allergens (Dermatophagoides pteronyssinus and/or farinae). All the patients suffered from severe recurrent dermatophytosis that responded poorly to antifungal treatment. The results of immunologic evaluation by laboratory tests were normal, except for a decrease in the ingestion phase by mononuclear phagocytes. After diagnosis of immunodeficiency, ketoconazole shampoo was used prophylactically and at the very first signs of recurrence of dermatophytosis, systemic antifungal treatment was started, without concurrent use of macrolides and with monitoring of hepatic function. The fungal infections responded well to this treatment and the patients' quality of life markedly improved.

Cytokine expression induced by Candida albicans in a model of cutaneous candidosis based on reconstituted human epidermis.

Skin equivalents based on reconstituted human epidermis have been used recently to establish models for allergic/irritant contact dermatitis and cutaneous candidosis. In the present study the cytokine expression pattern and the morphological alterations in experimental cutaneous candidosis were investigated by RT-PCR and histological analysis. In experimental cutaneous C albicans infection the mRNA expression levels of interleukin (IL)-1a, IL-1beta, IL-8, GM-CSF, Exodus-2, tumour necrosis factor-alpha and PSL (P-selectin ligand) were upregulated. Cytokine profile and histological features of infected skin (separation of keratinocytes, oedema, vacuolisation) were comparable to that seen in experimental contact dermatitis. These immunomodulatory and morphological similarities might reflect a common pathogenesis factor in both diseases.

Nasal vaccination induces the ability to eliminate Candida colonization without influencing the pre-existing antigen-specific IgE Abs: a possibility for the control of Candida-related atopic dermatitis.

In some cases of atopic dermatitis (AD), a possible pathological contribution to disease development by Candida albicans (C. albicans) has been suggested. AD patients with severe symptoms showing positive capsulated hydrolic carrier polymer radioallergosorbent test (CAP-RAST) against C. albicans demonstrated significantly higher levels of serum IgE Abs than did AD patients with mild symptoms. Based on the clinical facts, we have postulated that elimination of C. albicans by mucosal vaccination may lead to the restoration of severe symptoms in AD patients. For this purpose, we have developed an allergic murine model. Mice which were systemically challenged with C. albicans-associated antigen, manganese superoxide dismutase (MnSOD) or secreted aspartic proteases 2 (SAP2), together with alum, exhibited hyper IgE Abs. Systemically primed mice were then immunized with MnSOD or SAP2 plus cholera toxin (CT) as mucosal adjuvant through the nasal route. Interestingly, nasally immunized mice showed increased levels of Candida Ag-specific IgA Ab in fecal and nasal washes as well as in saliva samples but unchanged levels in Ag-specific IgE responses. Consistent with the Ab levels, high numbers of Candida Ag-specific IgA Ab-forming cells were induced in mononuclear cells isolated from intestinal lamina propria, nasal passages and salivary glands of nasally vaccinated mice with Ag plus CT. Furthermore, nasal immunization using MnSOD or SAP2 together with CT resulted in the elimination of colonized C. albicans from the intestinal tract. These results also suggest a potential role of mucosal vaccination in the control of C. albicans in patients with allergic diseases, including AD, although more research is needed to establish this therapeutic approach for mucosal vaccination.

Recombinant human antibody single chain variable fragments reactive with Candida albicans surface antigens.

A combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to identify phage clones capable of binding to the surface of Candida albicans blastoconidia. Single chain antibody variable fragments (scFv) derived from three clones detected C. albicans antigens by indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and Western blotting. The antigens detected were conserved among different strains of C. albicans and several other Candida species. Two scFv clones detected antigens specifically expressed by C. albicans blastoconidia; the third detected antigens in both blastoconidia and filamentous forms of C. albicans. The antigens containing the epitopes recognized by all three scFv could be extracted from blastoconidia by dithiothreitol, suggesting attachment to the cell wall via sulfhydryl bonds. Epitope detection by the scFv was sensitive to treatment of C. albicans blastoconidia with sodium periodate, but not proteinase K, indicating the cognate epitopes were composed of carbohydrate. Antigenic determinants for each of the three scFv were detected by immunohistochemical staining of skin sections from a model of cutaneous candidiasis, demonstrating expression in vivo. Through selection for the ability to bind intact organisms, the phage display system provides a means to rapidly identify monoclonal binding ligands to Candida surface antigens. Being entirely human, mature antibodies generated from the scFv have potential utility in the treatment of candidiasis.

Cutaneous candidosis in immunosuppressed patients.

One-hundred patients with candidosis of the skin, mucous membranes and nail plates (confirmed by cultivation) were examined. Topical or systemic antimycotic treatment was successful in 58 patients. After a complete evaluation, 42 patients were found to suffer from factors supporting candidosis--diabetes mellitus (12), anemia (3) and various local factors (10 patients); 27 patients showed a deficiency in cell-mediated immunity. In addition to intensive antimycotic therapy, successful treatment is affected by the actual immunity level and can be ensured by efficient immunomodulation treatment of immunodeficiency.