Rahul Kushwah

Complexity of dendritic cell subsets and their function in the host immune system

Dendritic cells (DCs) are professional antigen‐presenting cells that are critical for induction of adaptive immunity and tolerance. Traditionally DCs have been divided into two discrete subtypes, which comprise conventional and non‐conventional DCs. They are distributed across various organs in the body and comprise a heterogeneous population, which has been shown to display differences in terms of surface marker expression, function and origins. Recent studies have shed new light on the process of DC differentiation and distribution of DC subtypes in various organs. Although monocytes, macrophages and DCs share a common macrophage–DC progenitor, a common DC progenitor population has been identified that exclusively gives rise to DCs and not monocytes or macrophages. In this review, we discuss the recent advances in our understanding of DC differentiation and subtypes and provide a comprehensive overview of various DC subtypes with emphasis on their function and origins. Furthermore, in light of recent developments in the field of DC biology, we classify DCs based on the precursor populations from which the various DC subsets originate. We classify DCs derived from common DC progenitor and pre‐DC populations as conventional DCs, which includes both migratory and lymphoid‐resident DC subsets and classify monocyte‐derived DCs and plasmacytoid DCs as non‐conventional DCs.

Role of dendritic cells in the induction of regulatory T cells

Dendritic cells (DCs) play a key role in initiating immune responses and maintaining immune tolerance. In addition to playing a role in thymic selection, DCs play an active role in tolerance under steady state conditions through several mechanisms which are dependent on IL-10, TGF-β, retinoic acid, indoleamine-2,3,-dioxygenase along with vitamin D. Several of these mechanisms are employed by DCs in induction of regulatory T cells which are comprised of Tr1 regulatory T cells, natural and inducible foxp3+ regulatory T cells, Th3 regulatory T cells and double negative regulatory T cells. It appears that certain DC subsets are highly specialized in inducing regulatory T cell differentiation and in some tissues the local microenvironment plays a role in driving DCs towards a tolerogenic response. In this review we discuss the recent advances in our understanding of the mechanisms underlying DC driven regulatory T cell induction.

Dendritic cell apoptosis: regulation of tolerance versus immunity.

Dendritic cell (DC) apoptosis is an important event that regulates the balance between tolerance and immunity through multiple pathways, and defects in DC apoptosis can trigger autoimmunity. DC apoptosis is also associated with immunosuppression and has been observed under several pathologies and infections. Recent studies indicate that apoptotic DCs can also play an active role in induction of tolerance. This review discusses the regulatory pathways of DC apoptosis, stimuli inducing DC apoptosis, and the implications of DC apoptosis in the induction of immunosuppression and/or tolerance.

Uptake of apoptotic DC converts immature DC into tolerogenic DC that induce differentiation of Foxp3+ Treg

DC apoptosis has been observed in patients with cancer and sepsis, and defects in DC apoptosis have been implicated in the development of autoimmune diseases. However, the mechanisms of how DC apoptosis affects immune responses, are unclear. In this study, we showed that immature viable DC have the ability to uptake apoptotic DC as well as necrotic DC without it being recognized as an inflammatory event by immature viable DC. However, the specific uptake of apoptotic DC converted immature viable DC into tolerogenic DC, which were resistant to LPS‐induced maturation. These tolerogenic DC secreted increased levels of TGF‐β1, which induced differentiation of naïve T cells into Foxp3+ Treg. Furthermore, induction of Treg differentiation only occurred upon uptake of apoptotic DC and not apoptotic splenocytes by viable DC, indicating that it is specifically the uptake of apoptotic DC that gives viable immature DC the potential to induce Foxp3+ Treg. Taken together, these findings identify uptake of apoptotic DC by viable immature DC as an immunologically tolerogenic event.

Apoptotic Dendritic Cells Induce Tolerance in Mice through Suppression of Dendritic Cell Maturation and Induction of Antigen-Specific Regulatory T Cells

Dendritic cell (DC) apoptosis has been shown to play a role in maintaining a balance between tolerance and immunity. However, the mechanisms of how DC apoptosis affects the immune response are unclear. We have shown that in vitro culture of apoptotic DCs with immature DCs, results in their uptake by immature DCs, which subsequently turn into tolerogenic DCs, which then secrete TGF-β1 and induce Foxp3+ regulatory T cells (Tregs). In this study we looked at the effects of apoptotic DCs in vivo. Here we show that apoptotic DCs are taken up by viable DCs in vivo, which suppresses the ability of viable DCs to undergo maturation and subsequent migration to the lymph nodes in response to LPS. Additionally, delivery of apoptotic DCs to LPS inflamed lungs results in resolution of inflammation, which is mediated by the ability of apoptotic DCs to suppress response of viable DCs to LPS. Additionally, apoptotic DCs also induce TGF-β1 secretion in the mediastinal lymph nodes, which results in expansion of Foxp3+ Tregs. Most importantly, we show that delivery of apoptotic DCs followed by OVA in CFA to mice suppresses T cell response to OVA and instead induces de novo generation of OVA-specific Tregs. Furthermore, delivery of apoptotic DCs followed by OVA in CFA results in expansion of Tregs in TCR transgenic (OT-II) mice. These findings demonstrate that apoptotic DCs are taken up by viable DCs in vivo, which promotes tolerance through suppression of DC maturation and induction of Tregs.

Multiple roles of the epithelium-specific ETS transcription factor, ESE-1, in development and disease

The E26 transformation-specific (ETS) family of transcription factors comprises of 27 and 26 members in humans and mice, respectively, which are known to regulate many different biological processes, including cell proliferation, cell differentiation, embryonic development, neoplasia, hematopoiesis, angiogenesis, and inflammation. The epithelium-specific ETS transcription factor-1 (ESE-1) is a physiologically important ETS transcription factor, which has been shown to play a role in the pathogenesis of various diseases, and was originally characterized as having an epithelial-restricted expression pattern, thus placing it within the epithelium-specific ETS subfamily. Despite a large body of published work on ETS biology, much remains to be learned about the precise functions of ESE-1 and other epithelium-specific ETS factors in regulating diverse disease processes. Clues as to the specific function of ESE-1 in the setting of various diseases can be obtained from studies aimed at examining the expression of putative target genes regulated by ESE-1. Thus, this review will focus primarily on the various roles of ESE-1 in different pathophysiological processes, including regulation of epithelial cell differentiation during both intestinal development and lung regeneration; regulation of dendritic cell-driven T-cell differentiation during allergic airway inflammation; regulation of mammary gland development and breast cancer; and regulation of the effects of inflammatory stimuli within the setting of synovial joint and vascular inflammation. Understanding the exact mechanisms by which ESE-1 regulates these processes can have important implications for the treatment of a wide range of diseases.

Nacystelyn enhances adenoviral vector-mediated gene delivery to mouse airways

Adenoviral vector-mediated gene delivery has been vastly investigated for cystic fibrosis (CF) gene therapy; however, one of its drawbacks is the low efficiency of gene transfer, which is due to basolateral colocalization of viral receptors, immune responses to viral vectors and the presence of a thick mucus layer in the airways of CF patients. Therefore, enhancement of gene transfer can lead to reduction in the viral dosage, which could further reduce the acute toxicity associated with the use of adenoviral vectors. Nacystelyn (NAL) is a mucolytic agent with anti-inflammatory and antioxidant properties, and has been used clinically in CF patients to reduce mucus viscosity in the airways. In this study, we show that pretreatment of the airways with NAL followed by administration of adenoviral vectors in complex with DEAE-Dextran can significantly enhance gene delivery to the airways of mice without any harmful effects. Moreover, NAL pretreatment can reduce the airway inflammation, which is normally observed after delivery of adenoviral particles. Taken together, these results indicate that NAL pretreatment followed by adenoviral vector-mediated gene delivery can be beneficial to CF patients by increasing the efficiency of gene transfer to the airways, and reducing the acute toxicity associated with the administration of adenoviral vectors.

Elf3 plays a role in regulating bronchiolar epithelial repair kinetics following Clara cell-specific injury

E74-like transcription factor-3 (Elf3), a member of the E26 transformation-specific transcription factor family, is strongly expressed in epithelial-rich tissues, such as small intestine, fetal lung, and various lung cancers. Although previous studies have shown a defect in terminal differentiation of the small intestinal epithelium of Elf3-deficient (Elf3−/−) mice during embryonic development, very little is known about the role Elf3 may play in repair of the airway epithelium after injury. In order to investigate whether Elf3 is involved in regeneration of the bronchiolar epithelium after Clara cell-specific injury, we administered naphthalene to both wild-type (Elf3+/+) and Elf3−/− mice. Histopathological analysis revealed no significant difference in the extent of naphthalene-induced Clara cell necrosis between Elf3+/+ mice and Elf3−/− mice. In the bronchiolar epithelium of Elf3−/− mice, there was a substantial delay in the kinetics of cell proliferation and mitosis along with Clara cell renewal, whereas in the peribronchiolar interstitium, there was a significantly greater level of cell proliferation and mitosis in Elf3−/− mice than in Elf3+/+ mice. Last, the intensity of immunopositive signal for transforming growth factor-β type II receptor, which is a well-known transcriptional target gene of Elf3 and involved in the induction of epithelial cell differentiation, was significantly lower in the bronchiolar epithelium of Elf3−/− mice when compared with Elf3+/+ mice. Taken together, our results suggest that Elf3 plays an important role in the regulation of lung cell proliferation and differentiation during repair of the injured bronchiolar airway epithelium.

Characterization of Pulmonary T Cell Response to Helper-Dependent Adenoviral Vectors following Intranasal Delivery

In spite of the extensive research in the field of gene therapy, host immune responses continue to be the major barrier in translating basic research to clinical practice. Helper-dependent adenoviral (HD-Ad) vectors show great potential for pulmonary gene therapy, but the knowledge of pulmonary immune responses toward these vectors is very limited. In this study, we show that HD-Ad vectors are potent stimulators of dendritic cell (DC) maturation, thus leading to stimulation of T cell proliferation with ∼6% of naive CD4+ T cells from pulmonary mediastinal lymph node responding to HD-Ad-treated DCs. In contrast to the belief that HD-Ad vectors are unable to prime adaptive immune response, we show for the first time, through in vivo pulmonary studies in mice, that HD-Ad vectors can prime CD4+ and CD8+ T cell responses in the lung at high and substantially low doses. This indicates cross-presentation of HD-Ad-derived epitopes by DCs to prime CD8+ T cell responses. To assess the basis of pulmonary T cell response against HD-Ad vectors, we examined the response of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) in the lung. In response to HD-Ad delivery, there is induction of maturation in both cDC and pDC subsets, but it is the cDCs, not pDCs, that migrate rapidly to draining lymph nodes within the first 2 days after vector delivery to prime adaptive immune response against these vectors. These findings have implications for development of strategies to prevent adaptive immune responses against gene therapy vectors.

Elf3 Regulates Allergic Airway Inflammation by Controlling Dendritic Cell-Driven T Cell Differentiation

Elf3 belongs to the Ets family of transcription factors and has been implicated in inflammation. Elf3 is highly expressed in the lungs, and Elf3−/− mice are impaired in IL-6 production after intranasal LPS exposure. To identify the role of Elf3 in Th17-driven pulmonary inflammation, we have performed epicutaneous sensitization of Elf3−/− mice with OVA followed by airway OVA challenge and have identified Elf3−/− mice to be impaired in induction of Th17 response, attributable to impairment of IL-6 production by dendritic cells (DCs). However, increased serum levels of OVA-specific IgG1 and IgE were observed, pointing toward an exaggerated Th2 response. To study Th2 response, we performed i.p. sensitization of Elf3−/− mice with OVA and confirmed loss of Elf3 to result in an aggravated Th2 response, characterized by increased generation of IL-4–producing T cells, increased levels of OVA-specific IgE and IgG1 Ab titers, and increased serum levels of Th2 cytokines, together with extensive inflammation and mucus production in airways. Elf3−/− DCs were impaired in priming Th1 differentiation, which, in turn, promoted Th2 differentiation. This was mediated by the ability of Elf3−/− DCs to undergo hypermaturation but secrete significantly lower levels of IL-12 in response to inflammatory stimuli. The impairment of IL-12 production was due to impairment of IL-12p40 gene induction in Elf3−/− DCs in response to inflammatory stimuli. Taken together, our study identifies a novel function of Elf3 in regulating allergic airway inflammation by regulating DC-driven Th1, Th2, and Th17 differentiation.