The cellular architecture of the antimicrobial response network in human leprosy granulomas.

Granulomas are complex cellular structures composed predominantly of macrophages and lymphocytes that function to contain and kill invading pathogens. Here, we investigated the single-cell phenotypes associated with antimicrobial responses in human leprosy granulomas by applying single-cell and spatial sequencing to leprosy biopsy specimens. We focused on reversal reactions (RRs), a dynamic process whereby some patients with disseminated lepromatous leprosy (L-lep) transition toward self-limiting tuberculoid leprosy (T-lep), mounting effective antimicrobial responses. We identified a set of genes encoding proteins involved in antimicrobial responses that are differentially expressed in RR versus L-lep lesions and regulated by interferon-γ and interleukin-1ß. By integrating the spatial coordinates of the key cell types and antimicrobial gene expression in RR and T-lep lesions, we constructed a map revealing the organized architecture of granulomas depicting compositional and functional layers by which macrophages, T cells, keratinocytes and fibroblasts can each contribute to the antimicrobial response.

IL-1ß Induces the Rapid Secretion of the Antimicrobial Protein IL-26 from Th17 Cells.

Th17 cells play a critical role in the adaptive immune response against extracellular bacteria, and the possible mechanisms by which they can protect against infection are of particular interest. In this study, we describe, to our knowledge, a novel IL-1ß dependent pathway for secretion of the antimicrobial peptide IL-26 from human Th17 cells that is independent of and more rapid than classical TCR activation. We find that IL-26 is secreted 3 hours after treating PBMCs with Mycobacterium leprae as compared with 48 hours for IFN-γ and IL-17A. IL-1ß was required for microbial ligand induction of IL-26 and was sufficient to stimulate IL-26 release from Th17 cells. Only IL-1RI+ Th17 cells responded to IL-1ß, inducing an NF-κB-regulated transcriptome. Finally, supernatants from IL-1ß-treated memory T cells killed Escherichia coli in an IL-26-dependent manner. These results identify a mechanism by which human IL-1RI+ "antimicrobial Th17 cells" can be rapidly activated by IL-1ß as part of the innate immune response to produce IL-26 to kill extracellular bacteria.

TLR activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells.

Leprosy enables investigation of mechanisms by which the innate immune system contributes to host defense against infection, because in one form, the disease progresses, and in the other, the infection is limited. We report that Toll-like receptor (TLR) activation of human monocytes induces rapid differentiation into two distinct subsets: DC-SIGN+ CD16+ macrophages and CD1b+ DC-SIGN- dendritic cells. DC-SIGN+ phagocytic macrophages were expanded by TLR-mediated upregulation of interleukin (IL)-15 and IL-15 receptor. CD1b+ dendritic cells were expanded by TLR-mediated upregulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor, promoted T cell activation and secreted proinflammatory cytokines. Whereas DC-SIGN+ macrophages were detected in lesions and after TLR activation in all leprosy patients, CD1b+ dendritic cells were not detected in lesions or after TLR activation of peripheral monocytes in individuals with the progressive lepromatous form, except during reversal reactions in which bacilli were cleared by T helper type 1 (TH1) responses. In tuberculoid lepromatous lesions, DC-SIGN+ cells were positive for macrophage markers, but negative for dendritic cell markers. Thus, TLR-induced differentiation of monocytes into either macrophages or dendritic cells seems to crucially influence effective host defenses in human infectious disease.

Host-derived oxidized phospholipids and HDL regulate innate immunity in human leprosy.

Intracellular pathogens survive by evading the host immune system and accessing host metabolic pathways to obtain nutrients for their growth. Mycobacterium leprae, the causative agent of leprosy, is thought to be the mycobacterium most dependent on host metabolic pathways, including host-derived lipids. Although fatty acids and phospholipids accumulate in the lesions of individuals with the lepromatous (also known as disseminated) form of human leprosy (L-lep), the origin and significance of these lipids remains unclear. Here we show that in human L-lep lesions, there was preferential expression of host lipid metabolism genes, including a group of phospholipases, and that these genes were virtually absent from the mycobacterial genome. Host-derived oxidized phospholipids were detected in macrophages within L-lep lesions, and 1 specific oxidized phospholipid, 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine (PEIPC), accumulated in macrophages infected with live mycobacteria. Mycobacterial infection and host-derived oxidized phospholipids both inhibited innate immune responses, and this inhibition was reversed by the addition of normal HDL, a scavenger of oxidized phospholipids, but not by HDL from patients with L-lep. The accumulation of host-derived oxidized phospholipids in L-lep lesions is strikingly similar to observations in atherosclerosis, which suggests that the link between host lipid metabolism and innate immunity contributes to the pathogenesis of both microbial infection and metabolic disease.

New World cutaneous leishmaniasis: current challenges in diagnosis and parenteral treatment.

Many physicians in the United States and other nonendemic countries lack familiarity with New World cutaneous leishmaniasis (CL) and fail to include it in their differential diagnosis when seeing patients with suggestive lesions and recent high-risk travel. Moreover, even when the diagnosis of New World CL is considered and confirmed, physicians in the United States still face obstacles in obtaining appropriate treatment. In this report, we present 3 cases of New World CL that were either initially misdiagnosed or faced significant delays in therapy. We also discuss the optimal approach by which to confirm New World CL and to collaborate with professional colleagues at the Centers for Disease Control and Prevention in treating individual patients. In particular, when pentavalent antimonial treatment is needed for treatment, physicians must obtain appropriate diagnostic studies, communicate with experts at the Centers for Disease Control and Prevention, complete necessary paperwork, and obtain approval from their local institutional review board to administer it.

Activation and regulation of Toll-like receptors 2 and 1 in human leprosy.

The expression and activation of Toll-like receptors (TLRs) was investigated in leprosy, a spectral disease in which clinical manifestations correlate with the type of immune response mounted toward Mycobacterium leprae. TLR2-TLR1 heterodimers mediated cell activation by killed M. leprae, indicating the presence of triacylated lipoproteins. A genome-wide scan of M. leprae detected 31 putative lipoproteins. Synthetic lipopeptides representing the 19-kD and 33-kD lipoproteins activated both monocytes and dendritic cells. Activation was enhanced by type-1 cytokines and inhibited by type-2 cytokines. In addition, interferon (IFN)-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced TLR1 expression in monocytes and dendritic cells, respectively, whereas IL-4 downregulated TLR2 expression. TLR2 and TLR1 were more strongly expressed in lesions from the localized tuberculoid form (T-lep) as compared with the disseminated lepromatous form (L-lep) of the disease. These data provide evidence that regulated expression and activation of TLRs at the site of disease contribute to the host defense against microbial pathogens.

Mobile teledermatology: As doctors and patients are increasingly mobile, technology keeps us connected.

With advancements in electronics and health informatics, telemedicine has emerged as a cost-effective tool capable of increasing care to remote regions, facilitating specialist consults, supporting self-management by patients, and sharing knowledge over great distances. In this review, the authors discuss existing telemedicine modalities, highlight examples of mobile systems documented in the literature to date, and emphasize the data supporting the feasibility of telecommunication technologies to deliver dermatology services and education remotely. While many studies have suggested the potential for teledermatology to increase access to care in developing countries with few dermatologists, the authors share some of the most recent developments, including the use of diagnostic decision support software. The authors encourage a thriving and open network that will enhance the ongoing research and development of innovative and useful products. This network will also connect dermatologists willing to volunteer their consultation to health care workers in remote areas lacking specialists.

The cell fate regulator NUPR1 is induced by Mycobacterium leprae via type I interferon in human leprosy.

The initial interaction between a microbial pathogen and the host immune response influences the outcome of the battle between the host and the foreign invader. Leprosy, caused by the obligate intracellular pathogen Mycobacterium leprae, provides a model to study relevant human immune responses. Previous studies have adopted a targeted approach to investigate host response to M. leprae infection, focusing on the induction of specific molecules and pathways. By measuring the host transcriptome triggered by M. leprae infection of human macrophages, we were able to detect a host gene signature 24-48 hours after infection characterized by specific innate immune pathways involving the cell fate mechanisms autophagy and apoptosis. The top upstream regulator in the M. leprae-induced gene signature was NUPR1, which is found in the M. leprae-induced cell fate pathways. The induction of NUPR1 by M. leprae was dependent on the production of the type I interferon (IFN), IFN-ß. Furthermore, NUPR1 mRNA and protein were upregulated in the skin lesions from patients with the multibacillary form of leprosy. Together, these data indicate that M. leprae induces a cell fate program which includes NUPR1 as part of the host response in the progressive form of leprosy.

Human antimicrobial cytotoxic T lymphocytes, defined by NK receptors and antimicrobial proteins, kill intracellular bacteria.

Human CD8+ cytotoxic T lymphocytes (CTLs) contribute to antimicrobial defense against intracellular pathogens through secretion of cytotoxic granule proteins granzyme B, perforin, and granulysin. However, CTLs are heterogeneous in the expression of these proteins, and the subset(s) responsible for antimicrobial activity is unclear. Studying human leprosy, we found that the subset of CTLs coexpressing all three cytotoxic molecules is increased in the resistant form of the disease, can be expanded by interleukin-15 (IL-15), and is differentiated from naïve CD8+ T cells by Langerhans cells. RNA sequencing analysis identified that these CTLs express a gene signature that includes an array of surface receptors typically expressed by natural killer (NK) cells. We determined that CD8+ CTLs expressing granzyme B, perforin, and granulysin, as well as the activating NK receptor NKG2C, represent a population of "antimicrobial CTLs" (amCTLs) capable of T cell receptor (TCR)-dependent and TCR-independent release of cytotoxic granule proteins that mediate antimicrobial activity.

Activation of human CD4+ T cells by targeting MHC class II epitopes to endosomal compartments using human CD1 tail sequences.

Distinct CD4(+) T-cell epitopes within the same protein can be optimally processed and loaded into major histocompatibility complex (MHC) class II molecules in disparate endosomal compartments. The CD1 protein isoforms traffic to these same endosomal compartments as directed by unique cytoplasmic tail sequences, therefore we reasoned that antigen/CD1 chimeras containing the different CD1 cytoplasmic tail sequences could optimally target antigens to the MHC class II antigen presentation pathway. Evaluation of trafficking patterns revealed that all four human CD1-derived targeting sequences delivered antigen to the MHC class II antigen presentation pathway, to early/recycling, early/sorting and late endosomes/lysosomes. There was a preferential requirement for different CD1 targeting sequences for the optimal presentation of an MHC class II epitope in the following hierarchy: CD1b > CD1d = CD1c > > > CD1a or untargeted antigen. Therefore, the substitution of the CD1 ectodomain with heterologous proteins results in their traffic to distinct intracellular locations that intersect with MHC class II and this differential distribution leads to specific functional outcomes with respect to MHC class II antigen presentation. These findings may have implications in designing DNA vaccines, providing a greater variety of tools to generate T-cell responses against microbial pathogens or tumours.

Langerhans cells utilize CD1a and langerin to efficiently present nonpeptide antigens to T cells.

Langerhans cells (LCs) constitute a subset of DCs that initiate immune responses in skin. Using leprosy as a model, we investigated whether expression of CD1a and langerin, an LC-specific C-type lectin, imparts a specific functional role to LCs. LC-like DCs and freshly isolated epidermal LCs presented nonpeptide antigens of Mycobacterium leprae to T cell clones derived from a leprosy patient in a CD1a-restricted and langerin-dependent manner. LC-like DCs were more efficient at CD1a-restricted antigen presentation than monocyte-derived DCs. LCs in leprosy lesions coexpress CD1a and langerin, placing LCs in position to efficiently present a subset of antigens to T cells as part of the host response to human infectious disease.

Antigen-presenting cell candidates for HIV-1 transmission in human distal colonic mucosa defined by CD207 dendritic cells and CD209 macrophages.

A common route for HIV-1 infection is sexual transmission across colorectal mucosa, which is thought to be 10-2,000 times more vulnerable to infection than that of the female genital tract. Mucosal surfaces are the first line of defense against many pathogens but the antigen-presenting cells (APCs), key regulators of innate immunity and determinants of adaptive immunity, are not well defined in these target tissues. Using immunohistochemistry, dendritic cells expressing Langerin (CD207(+)), a lectin known to bind and internalize HIV-1, were detected in the periphery of colonic glands and sparsely scattered in the submucosa similarly in colorectal mucosa. This cell type, well known in skin, has generally not been reported in colonic/rectal mucosa. Unexpectedly, the largest APC population observed was a macrophage-like population expressing the well-characterized tissue macrophage markers CD68 and CD163. Confocal microscopy of these cells revealed colocalization of CD209 (DC-SIGN), a presumed dendritic cell marker believed to facilitate HIV-1 transmission, but not other dendritic cell markers. These results show evidence of the unconfirmed presence of Langerhans cells in colorectal mucosa and a predominance of macrophage-like APCs that express CD209 (DC-SIGN). These findings define potential target cells in the pathogenesis of HIV-1 transmission, which may have key implications for the study of early transmission events in normal colorectal mucosa, as well as other infectious diseases and primary immune diseases involving the gut.

NOD2 triggers an interleukin-32-dependent human dendritic cell program in leprosy.

It is unclear whether the ability of the innate immune system to recognize distinct ligands from a single microbial pathogen via multiple pattern recognition receptors (PRRs) triggers common pathways or differentially triggers specific host responses. In the human mycobacterial infection leprosy, we found that activation of monocytes via nucleotide-binding oligomerization domain-containing protein 2 (NOD2) by its ligand muramyl dipeptide, as compared to activation via heterodimeric Toll-like receptor 2 and Toll-like receptor 1 (TLR2/1) by triacylated lipopeptide, preferentially induced differentiation into dendritic cells (DCs), which was dependent on a previously unknown interleukin-32 (IL-32)-dependent mechanism. Notably, IL-32 was sufficient to induce monocytes to rapidly differentiate into DCs, which were more efficient than granulocyte-macrophage colony-stimulating factor (GM-CSF)-derived DCs in presenting antigen to major histocompatibility complex (MHC) class I-restricted CD8(+) T cells. Expression of NOD2 and IL-32 and the frequency of CD1b(+) DCs at the site of leprosy infection correlated with the clinical presentation; they were greater in patients with limited as compared to progressive disease. The addition of recombinant IL-32 restored NOD2-induced DC differentiation in patients with the progressive form of leprosy. In conclusion, the NOD2 ligand-induced, IL-32-dependent DC differentiation pathway contributes a key and specific mechanism for host defense against microbial infection in humans.

Human immunodeficiency virus and leishmaniasis.

The Leishmaniases are a group of diseases transmitted to humans by the bite of a sandfly, caused by protozoan parasites of the genus Leishmania. Various Leishmania species infect humans, producing a spectrum of clinical manifestations. It is estimated that 350 million people are at risk, with a global yearly incidence of 1-1.5 million for cutaneous and 500,000 for visceral Leishmaniasis (VL). VL is a major cause of morbidity and mortality in East Africa, Brazil and the Indian subcontinent. Co-infection with human immunodeficiency virus (HIV) alters the immune response to the disease. Here we review the immune response to Leishmania in the setting of HIV co-infection. Improved understanding of the immunology involved in co-infections may help in designing prophylactic and therapeutic strategies against Leishmaniasis.

Dendritic cell vaccination combined with CTLA4 blockade in patients with metastatic melanoma.

PURPOSE: Tumor antigen-loaded dendritic cells (DC) are believed to activate antitumor immunity by stimulating T cells, and CTL-associated antigen 4 (CTLA4)-blocking antibodies should release a key negative regulatory pathway on T cells. The combination was tested in a phase I clinical trial in patients with advanced melanoma. EXPERIMENTAL DESIGN: Autologous DC were pulsed with MART-1(26-35) peptide and administered with a dose escalation of the CTLA4-blocking antibody tremelimumab. Sixteen patients were accrued to five dose levels. Primary end points were safety and immune effects; clinical efficacy was a secondary end point. RESULTS: Dose-limiting toxicities of grade 3 diarrhea and grade 2 hypophysitis developed in two of three patients receiving tremelimumab at 10 mg/kg monthly. Four patients had an objective tumor response, two partial responses and two complete responses, all melanoma free between 2 and 4 years after study initiation. There was no difference in immune monitoring results between patients with an objective tumor response and those without a response. Exploratory gene expression analysis suggested that immune-related gene signatures, in particular for B-cell function, may be important in predicting response. CONCLUSION: The combination of MART-1 peptide-pulsed DC and tremelimumab results in objective and durable tumor responses at the higher range of the expected response rate with either agent alone.

Divergence of macrophage phagocytic and antimicrobial programs in leprosy.

Effective innate immunity against many microbial pathogens requires macrophage programs that upregulate phagocytosis and direct antimicrobial pathways, two functions generally assumed to be coordinately regulated. We investigated the regulation of these key functions in human blood-derived macrophages. Interleukin-10 (IL-10) induced the phagocytic pathway, including the C-type lectin CD209 and scavenger receptors, resulting in phagocytosis of mycobacteria and oxidized low-density lipoprotein. IL-15 induced the vitamin D-dependent antimicrobial pathway and CD209, yet the cells were less phagocytic. The differential regulation of macrophage functional programs was confirmed by analysis of leprosy lesions: the macrophage phagocytosis pathway was prominent in the clinically progressive, multibacillary form of the disease, whereas the vitamin D-dependent antimicrobial pathway predominated in the self-limited form and in patients undergoing reversal reactions from the multibacillary to the self-limited form. These data indicate that macrophage programs for phagocytosis and antimicrobial responses are distinct and differentially regulated in innate immunity to bacterial infections.

"Dermal dendritic cells" comprise two distinct populations: CD1+ dendritic cells and CD209+ macrophages.

A key cell type of the resident skin immune system is the dendritic cell (DC), which in normal skin is located in two distinct microanatomical compartments: Langerhans cells (LCs), mainly in the epidermis, and dermal DCs (DDCs), in the dermis. Here, the lineage of DDCs was investigated using monoclonal antibodies and immunohistology. We provide evidence that "DDC" comprise at least two major phenotypic populations of dendritic-appearing cells, immature DC expressing CD1, CD11c and CD208; and macrophages expressing CD209, CD206, CD163, and CD68. These data suggest that dermal dendritic-appearing macrophages comprise a novel part of the innate immune response in the resident skin immune system.

Regulation of human T-cell homing receptor expression in cutaneous bacterial infection.

We investigated the regulation of T-cell homing receptors in infectious disease by evaluating the cutaneous lymphocyte antigen (CLA) in human leprosy. We found that CLA-positive cells were enriched in the infectious lesions associated with restricting the growth of the pathogen Mycobacterium leprae, as assessed by the clinical course of infection. Moreover, CLA expression on T cells isolated from the peripheral blood of antigen-responsive tuberculoid leprosy patients increased in the presence of M. leprae (2.4-fold median increase; range 0.8-6.1, n = 17), but not in unresponsive lepromatous leprosy patients (1.0-fold median increase; range 0.1-2.2, n = 10; P < 0.005). Mycobacterium leprae specifically up-regulated the skin homing receptor, CLA, but not alpha(4)/beta(7), the intestinal homing receptor, which decreased on T cells of patients with tuberculoid leprosy after antigen stimulation (2.2-fold median decrease; range 1.6-3.4, n = 3). Our data indicate that CLA expression is regulated during the course of leprosy infection and suggest that T-cell responsiveness to a microbial antigen directs antigen-specific T cells to the site of infection.