Long-term tolerance to skin commensals is established neonatally through a specialized dendritic cell subgroup.

Early-life establishment of tolerance to commensal bacteria at barrier surfaces carries enduring implications for immune health but remains poorly understood. Here, we showed that tolerance in skin was controlled by microbial interaction with a specialized subset of antigen-presenting cells. More particularly, CD301b+ type 2 conventional dendritic cells (DCs) in neonatal skin were specifically capable of uptake and presentation of commensal antigens for the generation of regulatory T (Treg) cells. CD301b+ DC2 were enriched for phagocytosis and maturation programs, while also expressing tolerogenic markers. In both human and murine skin, these signatures were reinforced by microbial uptake. In contrast to their adult counterparts or other early-life DC subsets, neonatal CD301b+ DC2 highly expressed the retinoic-acid-producing enzyme, RALDH2, the deletion of which limited commensal-specific Treg cell generation. Thus, synergistic interactions between bacteria and a specialized DC subset critically support early-life tolerance at the cutaneous interface.

The best offense is a good beta-defensin.

The full range of receptors through which antimicrobial peptides exert their immunologic effects remains incompletely explored. Dong and colleagues identify Mgrpra2 as a G-coupled protein receptor on neutrophils, for which keratinocyte-derived Beta-defensins serve as key ligands. Binding of Mgrpra2 leads to release of neutrophil granules and Il-1ß, which helps shape skin microbiome composition and augments cutaneous defense against bacterial infection.

Baby's skin bacteria: first impressions are long-lasting.

Early life is a dynamic period for skin microbial colonization and immune development. We postulate that microbial exposures in this period durably alter the skin immune trajectory and later disease susceptibility. Bacteria contribute to infant skin immune imprinting via interactions with microbes as well as with cutaneous epithelial and immune cells. Excellent research is underway at the skin microbiome-immune interface, both in deciphering basic mechanisms and implementing their therapeutic applications. As emphasized herein, focusing on the unique opportunities and challenges presented by microbial immune modulation in early life will be important. In our view, only through dedicated study of skin-microbe crosstalk in this developmental window can we elucidate the molecular underpinnings of pivotal events that contribute to sustained host-microbe symbiosis.

Modification of the Pulmonary MyD88 Inflammatory Response Underlies the Role of the Yersinia pestis Pigmentation Locus in Primary Pneumonic Plague.

Pneumonic plague, caused by Yersinia pestis, is a rapidly progressing bronchopneumonia involving focal bacterial growth, neutrophilic congestion, and alveolar necrosis. Within a short time after inhalation of Y. pestis, inflammatory cytokines are expressed via the Toll/interleukin-1 (IL-1) adaptor myeloid differentiation primary response 88 (MyD88), which facilitates the primary lung infection. We previously showed that Y. pestis lacking the 102-kb chromosomal pigmentation locus (pgm) is unable to cause inflammatory damage in the lungs, whereas the wild-type (WT) strain induces the toxic MyD88 pulmonary inflammatory response. In this work, we investigated the involvement of the pgm in skewing the inflammatory response during pneumonic plague. We show that the early MyD88-dependent and -independent cytokine responses to pgm- Y. pestis infection of the lungs are similar yet distinct from those that occur during pgm+ infection. Furthermore, we found that MyD88 was necessary to prevent growth of the iron-starved pgm- Y. pestis despite the presence of iron chelators lactoferrin and transferrin. However, while this induced neutrophil recruitment, there was no hyperinflammatory response, and pulmonary disease was mild without MyD88. In contrast, growth in blood and tissues progressed rapidly in the absence of MyD88, due to an almost total loss of serum interferon gamma (IFN-γ). We further show that the expression of MyD88 by myeloid cells is important to control bacteremia but not the primary lung infection. The combined data indicate distinct roles for myeloid and nonmyeloid MyD88 and suggest that expression of the pgm is necessary to skew the inflammatory response in the lungs to cause pneumonic plague.

Skin Commensal Antigens: Taking the Road Less Traveled.

Although our knowledge of host-commensal interactions has increased exponentially, the mechanisms linking a specific commensal, its detection by the immune system, and its impact on tissue function are still often poorly understood. In a recent study in Cell, Linehan et al. dissect one of these interactions in the context of the skin, and demonstrate that Staphylococcus epidermidis antigens, presented through a non-classical pathway, drive the accumulation of CD8+ T cells that promote wound healing.

Multi-Immune Agonist Nanoparticle Therapy Stimulates Type I Interferons to Activate Antigen-Presenting Cells and Induce Antigen-Specific Antitumor Immunity.

Cancer immunity is mediated by a delicate orchestration between the innate and adaptive immune system both systemically and within the tumor microenvironment. Although several adaptive immunity molecular targets have been proven clinically efficacious, stand-alone innate immunity targeting agents have not been successful in the clinic. Here, we report a nanoparticle optimized for systemic administration that combines immune agonists for TLR9, STING, and RIG-I with a melanoma-specific peptide to induce antitumor immunity. These immune agonistic nanoparticles (iaNPs) significantly enhance the activation of antigen-presenting cells to orchestrate the development and response of melanoma-sensitized T-cells. iaNP treatment not only suppressed tumor growth in an orthotopic solid tumor model, but also significantly reduced tumor burden in a metastatic animal model. This combination biomaterial-based approach to coordinate innate and adaptive anticancer immunity provides further insights into the benefits of stimulating multiple activation pathways to promote tumor regression, while also offering an important platform to effectively and safely deliver combination immunotherapies for cancer.

Yersinia pestis Exploits Early Activation of MyD88 for Growth in the Lungs during Pneumonic Plague.

Yersinia pestis causes bubonic, pneumonic, and septicemic plague. Although no longer responsible for pandemic outbreaks, pneumonic plague continues to be a challenge for medical treatment and has been classified as a reemerging disease in some parts of the world. In the early stage of infection, inflammatory responses are believed to be suppressed by Y. pestis virulence factors in order to prevent clearance, while later, the hyperactivation of inflammation contributes to the progression of disease. In this work, we sought to identify the host factors that mediate this process and studied the role of the Toll/interleukin 1 (IL-1) receptor adapter and major inflammatory mediator myeloid differentiation primary response 88 (MyD88) in pneumonic plague. We show that pulmonary challenge of Myd88-/- mice with wild-type (WT) Y. pestis results in significant loss of pro- and anti-inflammatory cytokines and chemokines, especially gamma interferon (IFN-γ) and KC, in the lungs compared to that in WT mice. Bacterial growth in the lungs occurred more rapidly in the WT mice, however, indicating a role for the MyD88 response in facilitating the primary lung infection. Nevertheless, Myd88-/- mice were more sensitive to lethality from secondary septicemic plague. Together these findings indicate a central role for MyD88 during the biphasic inflammatory response to pulmonary Y. pestis infection. In the early phase, low-level MyD88-dependent chemokine expression limits initial growth but facilitates Y. pestis access to a protected replicative niche. The later hyperinflammatory phase is partially MyD88 dependent and ineffective in the lungs but controls systemic infection and reduces the progression of secondary septicemic plague.

Induction of Type I Interferon through a Noncanonical Toll-Like Receptor 7 Pathway during Yersinia pestis Infection.

Yersinia pestis causes bubonic, pneumonic, and septicemic plague, diseases that are rapidly lethal to most mammals, including humans. Plague develops as a consequence of bacterial neutralization of the host's innate immune response, which permits uncontrolled growth and causes the systemic hyperactivation of the inflammatory response. We previously found that host type I interferon (IFN) signaling is induced during Y. pestis infection and contributes to neutrophil depletion and disease. In this work, we show that type I IFN expression is derived from the recognition of intracellular Y. pestis by host Toll-like receptor 7 (TLR7). Type I IFN expression proceeded independent of myeloid differentiation factor 88 (MyD88), which is the only known signaling adaptor for TLR7, suggesting that a noncanonical mechanism occurs in Y. pestis-infected macrophages. In the murine plague model, TLR7 was a significant contributor to the expression of serum IFN-ß, whereas MyD88 was not. Furthermore, like the type I IFN response, TLR7 contributed to the lethality of septicemic plague and was associated with the suppression of neutrophilic inflammation. In contrast, TLR7 was important for defense against disease in the lungs. Together, these data demonstrate that an atypical TLR7 signaling pathway contributes to type I IFN expression during Y. pestis infection and suggest that the TLR7-driven type I IFN response plays an important role in determining the outcome of plague.

Early apoptosis of macrophages modulated by injection of Yersinia pestis YopK promotes progression of primary pneumonic plague.

Yersinia pestis causes pneumonic plague, a disease characterized by inflammation, necrosis and rapid bacterial growth which together cause acute lung congestion and lethality. The bacterial type III secretion system (T3SS) injects 7 effector proteins into host cells and their combined activities are necessary to establish infection. Y. pestis infection of the lungs proceeds as a biphasic inflammatory response believed to be regulated through the control of apoptosis and pyroptosis by a single, well-conserved T3SS effector protein YopJ. Recently, YopJ-mediated pyroptosis, which proceeds via the NLRP3-inflammasome, was shown to be regulated by a second T3SS effector protein YopK in the related strain Y. pseudotuberculosis. In this work, we show that for Y. pestis, YopK appears to regulate YopJ-mediated apoptosis, rather than pyroptosis, of macrophages. Inhibition of caspase-8 blocked YopK-dependent apoptosis, suggesting the involvement of the extrinsic pathway, and appeared cell-type specific. However, in contrast to yopJ, deletion of yopK caused a large decrease in virulence in a mouse pneumonic plague model. YopK-dependent modulation of macrophage apoptosis was observed at 6 and 24 hours post-infection (HPI). When YopK was absent, decreased populations of macrophages and dendritic cells were seen in the lungs at 24 HPI and correlated with resolution rather than progression of inflammation. Together the data suggest that Y. pestis YopK may coordinate the inflammatory response during pneumonic plague through the regulation of apoptosis of immune cells.

FLG Deficiency in Mice Alters the Early-Life CD4+ T-Cell Response to Skin Commensal Bacteria.

FLG variants underlie ichthyosis vulgaris and increased risk of atopic dermatitis, conditions typified by disruption of the skin microbiome and cutaneous immune response. Yet, it remains unclear whether neonatal skin barrier compromise because of FLG deficiency alters the quality of commensal-specific T cells and the functional impact of such responses. To address these questions, we profiled changes in the skin barrier and early cutaneous immune response of neonatal C57BL/6 Flg‒/‒ and wild-type mice using single-cell RNA sequencing, flow cytometry, and other modalities. Flg‒/‒ neonates showed little alteration in transepidermal water loss or lipid- or corneocyte-related gene expression. However, they showed increases in barrier disruption genes, epidermal dye penetration, and numbers of skin CD4+ T cells. Using an engineered strain of Staphylococcus epidermidis (S. epidermidis 2W) to study the response to neonatal skin colonization, we found that commensal-specific CD4+ T cells were skewed in Flg‒/‒ pups toward effector rather than regulatory T cells. This altered response persisted into adulthood, where it was typified by T helper 17 (Th17) cells and associated with increased susceptibility to imiquimod-induced skin inflammation. Thus, subtle but impactful differences in neonatal barrier function in Flg‒/‒ mice are accompanied by a skewed commensal-specific CD4+ response, with enduring consequences for skin immune homeostasis.

Commensal myeloid crosstalk in neonatal skin regulates long-term cutaneous type 17 inflammation.

Early life microbe-immune interactions at barrier surfaces have lasting impacts on the trajectory towards health versus disease. Monocytes, macrophages and dendritic cells are primary sentinels in barrier tissues, yet the salient contributions of commensal-myeloid crosstalk during tissue development remain poorly understood. Here, we identify that commensal microbes facilitate accumulation of a population of monocytes in neonatal skin. Transient postnatal depletion of these monocytes resulted in heightened IL-17A production by skin T cells, which was particularly sustained among CD4+ T cells into adulthood and sufficient to exacerbate inflammatory skin pathologies. Neonatal skin monocytes were enriched in expression of negative regulators of the IL-1 pathway. Functional in vivo experiments confirmed a key role for excessive IL-1R1 signaling in T cells as contributing to the dysregulated type 17 response in neonatal monocyte-depleted mice. Thus, a commensal-driven wave of monocytes into neonatal skin critically facilitates long-term immune homeostasis in this prominent barrier tissue.

Intestinal inflammation alters the antigen-specific immune response to a skin commensal.

Resident microbes in skin and gut predominantly impact local immune cell function during homeostasis. However, colitis-associated neutrophilic skin disorders suggest possible breakdown of this compartmentalization with disease. Using a model wherein neonatal skin colonization by Staphylococcus epidermidis facilitates generation of commensal-specific tolerance and CD4+ regulatory T cells (Tregs), we ask whether this response is perturbed by gut inflammation. Chemically induced colitis is accompanied by intestinal expansion of S. epidermidis and reduces gut-draining lymph node (dLN) commensal-specific Tregs. It also results in reduced commensal-specific Tregs in skin and skin-dLNs and increased skin neutrophils. Increased CD4+ circulation between gut and skin dLN suggests that the altered cutaneous response is initiated in the colon, and resistance to colitis-induced effects in Cd4creIl1r1fl/fl mice implicate interleukin (IL)-1 in mediating the altered commensal-specific response. These findings provide mechanistic insight into observed connections between inflammatory skin and intestinal diseases.

Regulatory T cells promote innate inflammation after skin barrier breach via TGF-ß activation.

Regulatory T cells (Tregs) use multiple mechanisms to attenuate inflammation and prevent autoimmunity. Tregs residing in peripheral (i.e., nonlymphoid) tissues have specialized functions; specifically, skin Tregs promote wound healing, suppress dermal fibrosis, facilitate epidermal regeneration, and augment hair follicle cycling. Here, we demonstrated that skin Tregs were transcriptionally attuned to interact with their tissue environment through increased expression of integrin and TGF-ß pathway genes that influence epithelial cell biology. We identified a molecular pathway where skin Tregs license keratinocytes to promote innate inflammation after skin barrier breach. Using a single-cell discovery approach, we identified preferential expression of the integrin αvß8 on skin Tregs Upon skin injury, Tregs used this integrin to activate latent TGF-ß, which acted directly on epithelial cells to promote CXCL5 production and neutrophil recruitment. Induction of this circuit delayed epidermal regeneration but provided protection from Staphylococcus aureus infection across a compromised barrier. Thus, αvß8-expressing Tregs in the skin, somewhat paradoxical to their canonical immunosuppressive functions, facilitated inflammation acutely after loss of barrier integrity to promote host defense against infection.

Developing Human Skin Contains Lymphocytes Demonstrating a Memory Signature.

Lymphocytes in barrier tissues play critical roles in host defense and homeostasis. These cells take up residence in tissues during defined developmental windows, when they may demonstrate distinct phenotypes and functions. Here, we utilized mass and flow cytometry to elucidate early features of human skin immunity. Although most conventional αß T (Tconv) cells in fetal skin have a naive, proliferative phenotype, a subset of CD4+ Tconv and CD8+ cells demonstrate memory-like features and a propensity for interferon (IFN)γ production. Skin regulatory T cells dynamically accumulate over the second trimester in temporal and regional association with hair follicle development. These fetal skin regulatory T cells (Tregs) demonstrate an effector memory phenotype while differing from their adult counterparts in expression of key effector molecules. Thus, we identify features of prenatal skin lymphocytes that may have key implications for understanding antigen and allergen encounters in utero and in infancy.

Toxin-Triggered Interleukin-1 Receptor Signaling Enables Early-Life Discrimination of Pathogenic versus Commensal Skin Bacteria.

The host must develop tolerance to commensal microbes and protective responses to infectious pathogens, yet the mechanisms enabling a privileged relationship with commensals remain largely unknown. Skin colonization by commensal Staphylococcus epidermidis facilitates immune tolerance preferentially in neonates via induction of antigen-specific regulatory T cells (Tregs). Here, we demonstrate that this tolerance is not indiscriminately extended to all bacteria encountered in this early window. Rather, neonatal colonization by Staphylococcus aureus minimally enriches for antigen-specific Tregs and does not prevent skin inflammation upon later-life exposure. S. aureus α-toxin contributes to this response by stimulating myeloid cell production of IL-1ß, which limits S. aureus-specific Tregs. Loss of α-toxin or the IL-1 receptor increases Treg enrichment, whereas topical application of IL-1ß or α-toxin diminishes tolerogenic responses to S. epidermidis. Thus, the preferential activation of a key alarmin pathway facilitates early discrimination of microbial "foe" from "friend," thereby preventing tolerance to a common skin pathogen.

Bacterial programming of host responses: coordination between type I interferon and cell death.

During mammalian infection, bacteria induce cell death from an extracellular or intracellular niche that can protect or hurt the host. Data is accumulating that associate type I interferon (IFN) signaling activated by intracellular bacteria with programmed death of immune effector cells and enhanced virulence. Multiple pathways leading to IFN-dependent host cell death have been described, and in some cases it is becoming clear how these mechanisms contribute to virulence. Yet common mechanisms of IFN-enhanced bacterial pathogenesis are not obvious and no specific interferon stimulated genes have yet been identified that cause sensitivity to pathogen-induced cell death. In this review, we will summarize some bacterial infections caused by facultative intracellular pathogens and what is known about how type I IFN signaling may promote the replication of extracellular bacteria rather than stimulate protection. Each of these pathogens can survive phagocytosis but their intracellular life cycles are very different, they express distinct virulence factors and trigger different pathways of immune activation and crosstalk. These differences likely lead to widely varying amounts of type I IFN expression and a different inflammatory environment, but these may not be important to the pathologic effects on the host. Instead, each pathogen induces programmed cell death of key immune cells that have been sensitized by the activation of the type I IFN response. We will discuss how IFN-dependent host cell death may increase host susceptibility and try to understand common pathways of pathogenesis that lead to IFN-enhanced bacterial virulence.

Host stress and immune responses during aerosol challenge of Brown Norway rats with Yersinia pestis.

Inhalation exposure models are becoming the preferred method for the comparative study of respiratory infectious diseases due to their resemblance to the natural route of infection. To enable precise delivery of pathogen to the lower respiratory tract in a manner that imposes minimal biosafety risk, nose-only exposure systems have been developed. Early inhalation exposure technology for infectious disease research grew out of technology used in asthma research where predominantly the Collison nebulizer is used to generate an aerosol by beating a liquid sample against glass. Although infectious aerosol droplets of 1-5 µm in size can be generated, the Collison often causes loss of viability. In this work, we evaluate a gentler method for aerosolization of living cells and describe the use of the Sparging Liquid Aerosol Generator (SLAG) in a rat pneumonic plague model. The SLAG creates aerosols by continuous dripping of liquid sample on a porous metal disc. We show the generation of 0.5-1 µm Yersinia pestis aerosol particles using the SLAG with spray factors typically ranging from 10(-7) to 10(-8) with no detectable loss of bacterial viability. Delivery of these infectious particles via nose-only exposure led to the rapid development of lethal pneumonic plague. Further, we evaluated the effect of restraint-stress imposed by the nose-only exposure chamber on early inflammatory responses and bacterial deposition. Elevated serum corticosterone which peaked at 2 h post-procedure indicated the animals experienced stress as a result of restraint in the nose-only chamber. However, we observed no correlation between elevated corticosterone and the amount of bacterial deposition or inflammation in the lungs. Together these data demonstrate the utility of the SLAG and the nose-only chamber for aerosol challenge of rodents by Y. pestis.