Alloreactive memory CD4 T cells promote transplant rejection by engaging DCs to induce innate inflammation and CD8 T cell priming.

Alloreactive memory T cells have been implicated as central drivers of transplant rejection. Perplexingly, innate cytokines, such as IL-6, IL-1ß, and IL-12, are also associated with rejection of organ transplants. However, the pathways of innate immune activation in allogeneic transplantation are unclear. While the role of microbial and cell death products has been previously described, we identified alloreactive memory CD4 T cells as the primary triggers of innate inflammation. Memory CD4 T cells engaged MHC II-mismatched dendritic cells (DCs), leading to the production of innate inflammatory cytokines. This innate inflammation was independent of several pattern recognition receptors and was primarily driven by TNF superfamily ligands expressed by alloreactive memory CD4 T cells. Blocking of CD40L and TNFα resulted in dampened inflammation, and mice genetically deficient in these molecules exhibited prolonged survival of cardiac allografts. Furthermore, myeloid cell and CD8 T cell infiltration into cardiac transplants was compromised in both CD40L- and TNFα-deficient recipients. Strikingly, we found that priming of naive alloreactive CD8 T cells was dependent on licensing of DCs by memory CD4 T cells. This study unravels the key mechanisms by which alloreactive memory CD4 T cells contribute to destructive pathology and transplant rejection.

Control of adaptive immunity by pattern recognition receptors.

One of the most significant conceptual advances in immunology in recent history is the recognition that signals from the innate immune system are required for induction of adaptive immune responses. Two breakthroughs were critical in establishing this paradigm: the identification of dendritic cells (DCs) as the cellular link between innate and adaptive immunity and the discovery of pattern recognition receptors (PRRs) as a molecular link that controls innate immune activation as well as DC function. Here, we recount the key events leading to these discoveries and discuss our current understanding of how PRRs shape adaptive immune responses, both indirectly through control of DC function and directly through control of lymphocyte function. In this context, we provide a conceptual framework for how variation in the signals generated by PRR activation, in DCs or other cell types, can influence T cell differentiation and shape the ensuing adaptive immune response.

Effector memory T cells induce innate inflammation by triggering DNA damage and a non-canonical STING pathway in dendritic cells.

Cognate interaction between CD4+ effector memory T (TEM) cells and dendritic cells (DCs) induces innate inflammatory cytokine production, resulting in detrimental autoimmune pathology and cytokine storms. While TEM cells use tumor necrosis factor (TNF) superfamily ligands to activate DCs, whether TEM cells prompt other DC-intrinsic changes that influence the innate inflammatory response has never been investigated. We report the surprising discovery that TEM cells trigger double-strand DNA breaks via mitochondrial reactive oxygen species (ROS) production in interacting DCs. Initiation of the DNA damage response in DCs induces activation of a cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-independent, non-canonical stimulator of interferon genes (STING)-TNF receptor-associated factor 6 (TRAF6)-nuclear factor κB (NF-κB) signaling axis. Consequently, STING-deficient DCs display reduced NF-κB activation and subsequent defects in transcriptional induction and functional production of interleukin-1ß (IL-1ß) and IL-6 following their interaction with TEM cells. The discovery of TEM cell-induced innate inflammation through DNA damage and a non-canonical STING-NF-κB pathway presents this pathway as a potential target to alleviate T cell-driven inflammation in autoimmunity and cytokine storms.

Immune checkpoint blockade induces gut microbiota translocation that augments extraintestinal antitumor immunity.

Gut microbiota, specifically gut bacteria, are critical for effective immune checkpoint blockade therapy (ICT) for cancer. The mechanisms by which gut microbiota augment extraintestinal anticancer immune responses, however, are largely unknown. Here, we find that ICT induces the translocation of specific endogenous gut bacteria into secondary lymphoid organs and subcutaneous melanoma tumors. Mechanistically, ICT induces lymph node remodeling and dendritic cell (DC) activation, which facilitates the translocation of a selective subset of gut bacteria to extraintestinal tissues to promote optimal antitumor T cell responses in both the tumor-draining lymph nodes (TDLNs) and the primary tumor. Antibiotic treatment results in decreased gut microbiota translocation into mesenteric lymph nodes (MLNs) and TDLNs, diminished DC and effector CD8+ T cell responses, and attenuated responses to ICT. Our findings illuminate a key mechanism by which gut microbiota promote extraintestinal anticancer immunity.

IEC-intrinsic IL-1R signaling holds dual roles in regulating intestinal homeostasis and inflammation.

Intestinal epithelial cells (IECs) constitute a critical first line of defense against microbes. While IECs are known to respond to various microbial signals, the precise upstream cues regulating diverse IEC responses are not clear. Here, we discover a dual role for IEC-intrinsic interleukin-1 receptor (IL-1R) signaling in regulating intestinal homeostasis and inflammation. Absence of IL-1R in epithelial cells abrogates a homeostatic antimicrobial program including production of antimicrobial peptides (AMPs). Mice deficient for IEC-intrinsic IL-1R are unable to clear Citrobacter rodentium (C. rodentium) but are protected from DSS-induced colitis. Mechanistically, IL-1R signaling enhances IL-22R-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in IECs leading to elevated production of AMPs. IL-1R signaling in IECs also directly induces expression of chemokines as well as genes involved in the production of reactive oxygen species. Our findings establish a protective role for IEC-intrinsic IL-1R signaling in combating infections but a detrimental role during colitis induced by epithelial damage.

Farnesoid X receptor antagonizes macrophage-dependent licensing of effector T lymphocytes and progression of sclerosing cholangitis.

Immune-mediated bile duct epithelial injury and toxicity of retained hydrophobic bile acids drive disease progression in fibrosing cholangiopathies such as biliary atresia or primary sclerosing cholangitis. Emerging therapies include pharmacological agonists to farnesoid X receptor (FXR), the master regulator of hepatic synthesis, excretion, and intestinal reuptake of bile acids. Unraveling the mechanisms of action of pharmacological FXR agonists in the treatment of sclerosing cholangitis (SC), we found that intestinally restricted FXR activation effectively reduced bile acid pool size but did not improve the SC phenotype in MDR2-/- mice. In contrast, systemic FXR activation not only lowered bile acid synthesis but also suppressed proinflammatory cytokine production by liver-infiltrating inflammatory cells and blocked progression of hepatobiliary injury. The hepatoprotective activity was linked to suppressed production of IL1ß and TNFα by hepatic macrophages and inhibition of TH1/TH17 lymphocyte polarization. Deletion of FXR in myeloid cells caused aberrant TH1 and TH17 lymphocyte responses in diethoxycarbonyl-1,4-dihydrocollidine-induced SC and rendered these mice resistant to the anti-inflammatory and liver protective effects of systemic FXR agonist treatment. Pharmacological FXR activation reduced IL1ß and IFNγ production by liver- and blood-derived mononuclear cells from patients with fibrosing cholangiopathies. In conclusion, we demonstrate FXR to control the macrophage-TH1/17 axis, which is critically important for the progression of SC. Hepatic macrophages are cellular targets of systemic FXR agonist therapy for cholestatic liver disease.

Effector memory CD4+ T cells induce damaging innate inflammation and autoimmune pathology by engaging CD40 and TNFR on myeloid cells.

Cytokine storm and sterile inflammation are common features of T cell-mediated autoimmune diseases and T cell-targeted cancer immunotherapies. Although blocking individual cytokines can mitigate some pathology, the upstream mechanisms governing overabundant innate inflammatory cytokine production remain unknown. Here, we have identified a critical signaling node that is engaged by effector memory T cells (TEM) to mobilize a broad proinflammatory program in the innate immune system. Cognate interactions between TEM and myeloid cells led to induction of an inflammatory transcriptional profile that was reminiscent, yet entirely independent, of classical pattern recognition receptor (PRR) activation. This PRR-independent "de novo" inflammation was driven by preexisting TEM engagement of both CD40 and tumor necrosis factor receptor (TNFR) on myeloid cells. Cytokine toxicity and autoimmune pathology could be completely rescued by ablating these pathways genetically or pharmacologically in multiple models of T cell-driven inflammation, indicating that TEM instruction of the innate immune system is a primary driver of associated immunopathology. Thus, we have identified a previously unknown trigger of cytokine storm and autoimmune pathology that is amenable to therapeutic interventions.

Environmental allergens trigger type 2 inflammation through ripoptosome activation.

Environmental allergens, including fungi, insects and mites, trigger type 2 immunity; however, the innate sensing mechanisms and initial signaling events remain unclear. Herein, we demonstrate that allergens trigger RIPK1-caspase 8 ripoptosome activation in epithelial cells. The active caspase 8 subsequently engages caspases 3 and 7, which directly mediate intracellular maturation and release of IL-33, a pro-atopy, innate immunity, alarmin cytokine. Mature IL-33 maintained functional interaction with the cognate ST2 receptor and elicited potent pro-atopy inflammatory activity in vitro and in vivo. Inhibiting caspase 8 pharmacologically and deleting murine Il33 and Casp8 each attenuated allergic inflammation in vivo. Clinical data substantiated ripoptosome activation and IL-33 maturation as likely contributors to human allergic inflammation. Our findings reveal an epithelial barrier, allergen-sensing mechanism that converges on the ripoptosome as an intracellular molecular signaling platform, triggering type 2 innate immune responses. These findings have significant implications for understanding and treating human allergic diseases.

Innate control of adaptive immunity and adaptive instruction of innate immunity: bi-directional flow of information.

The ability of the innate and adaptive immune systems to communicate with each other is central to protective immune responses and maintenance of host health. Myeloid cells of the innate immune system are able to sense microbial ligands, perturbations in cellular homeostasis, and virulence factors, thereby allowing them to relay distinct pathogen-specific information to naïve T cells in the form of pathogen-derived peptides and a unique cytokine milieu. Once primed, effector T helper cells produce lineage-defining cytokines to help combat the original pathogen, and a subset of these cells persist as memory or effector-memory populations. These memory T cells then play a dual role in host protection by not only responding rapidly to reinfection, but by also directly instructing myeloid cells to express licensing cytokines. This means there is a bi-directional flow of information first from the innate to the adaptive immune system, and then from the adaptive back to innate immune system. Here, we focus on how signals, first from pathogens and then from primed effector and memory T cells, are integrated by myeloid cells and its consequences for protective immunity or systemic inflammation.

IRF1 governs the differential interferon-stimulated gene responses in human monocytes and macrophages by regulating chromatin accessibility.

Myeloid lineage cells use TLRs to recognize and respond to diverse microbial ligands. Although unique transcription factors dictate the outcome of specific TLR signaling, whether lineage-specific differences exist to further modulate the quality of TLR-induced inflammation remains unclear. Comprehensive analysis of global gene transcription in human monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells stimulated with various TLR ligands identifies multiple lineage-specific, TLR-responsive gene programs. Monocytes are hyperresponsive to TLR7/8 stimulation that correlates with the higher expression of the receptors. While macrophages and monocytes express similar levels of TLR4, macrophages, but not monocytes, upregulate interferon-stimulated genes (ISGs) in response to TLR4 stimulation. We find that TLR4 signaling in macrophages uniquely engages transcription factor IRF1, which facilitates the opening of ISG loci for transcription. This study provides a critical mechanistic basis for lineage-specific TLR responses and uncovers IRF1 as a master regulator for the ISG transcriptional program in human macrophages.

Deep sequencing reveals a DAP1 regulatory haplotype that potentiates autoimmunity in systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is a clinically heterogeneous autoimmune disease characterized by the development of anti-nuclear antibodies. Susceptibility to SLE is multifactorial, with a combination of genetic and environmental risk factors contributing to disease development. Like other polygenic diseases, a significant proportion of estimated SLE heritability is not accounted for by common disease alleles analyzed by SNP array-based GWASs. Death-associated protein 1 (DAP1) was implicated as a candidate gene in a previous familial linkage study of SLE and rheumatoid arthritis, but the association has not been explored further. RESULTS: We perform deep sequencing across the DAP1 genomic segment in 2032 SLE patients, and healthy controls, and discover a low-frequency functional haplotype strongly associated with SLE risk in multiple ethnicities. We find multiple cis-eQTLs embedded in a risk haplotype that progressively downregulates DAP1 transcription in immune cells. Decreased DAP1 transcription results in reduced DAP1 protein in peripheral blood mononuclear cells, monocytes, and lymphoblastoid cell lines, leading to enhanced autophagic flux in immune cells expressing the DAP1 risk haplotype. Patients with DAP1 risk allele exhibit significantly higher autoantibody titers and altered expression of the immune system, autophagy, and apoptosis pathway transcripts, indicating that the DAP1 risk allele mediates enhanced autophagy, leading to the survival of autoreactive lymphocytes and increased autoantibody. CONCLUSIONS: We demonstrate how targeted sequencing captures low-frequency functional risk alleles that are missed by SNP array-based studies. SLE patients with the DAP1 genotype have distinct autoantibody and transcription profiles, supporting the dissection of SLE heterogeneity by genetic analysis.

TLR signaling adapter BCAP regulates inflammatory to reparatory macrophage transition by promoting histone lactylation.

Macrophages respond to microbial ligands and various noxious cues by initiating an inflammatory response aimed at eliminating the original pathogenic insult. Transition of macrophages from a proinflammatory state to a reparative state, however, is vital for resolution of inflammation and return to homeostasis. The molecular players governing this transition remain poorly defined. Here, we find that the reparative macrophage transition is dictated by B-cell adapter for PI3K (BCAP). Mice harboring a macrophage-specific deletion of BCAP fail to recover from and succumb to dextran sulfate sodium-induced colitis due to prolonged intestinal inflammation and impaired tissue repair. Following microbial stimulation, gene expression in WT macrophages switches from an early inflammatory signature to a late reparative signature, a process that is hampered in BCAP-deficient macrophages. We find that absence of BCAP hinders inactivation of FOXO1 and GSK3ß, which contributes to their enhanced inflammatory state. BCAP deficiency also results in defective aerobic glycolysis and reduced lactate production. This translates into reduced histone lactylation and decreased expression of reparative macrophage genes. Thus, our results reveal BCAP to be a critical cell-intrinsic switch that regulates transition of inflammatory macrophages to reparative macrophages by imprinting epigenetic changes.

Hypersensitivity of Vps33B mutant flies to non-pathogenic infections is dictated by aberrant activation of p38b MAP kinase.

Loss of the arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome-linked Vps33B protein results in exaggerated inflammatory responses upon activation of receptors of the innate immune system in both vertebrates and flies. However, little is known about the signaling elements downstream of these receptors that are critical for the hypersensitivity of Vps33B mutants. Here, we show that p38b MAP kinase contributes to the enhanced inflammatory responses in flies lacking Vps33B. Loss of p38b mitogen-activated protein kinase (MAPK) reduces enhanced inflammatory responses and prolongs the survival of infected Vps33B deficient flies. The function of p38 MAPK is not limited to its proinflammatory effects downstream of the PGRP-LC receptor as p38 also modulates endosomal trafficking of PGRP-LC and phagocytosis of bacteria. Expression of constitutively active p38b MAPK, but not dominant negative p38b MAPK enhances accumulation of endocytosed PGRP-LC receptors or phagocytosed bacteria within cells. Moreover, p38 MAPK is required for induction of macropinocytosis, an alternate pathway for the downregulation of immune receptors. Together, our data indicate that p38 MAPK activates multiple pathways that can contribute to the dysregulation of innate immune signaling in ARC syndrome.

Suppression of Inflammasome Activation by IRF8 and IRF4 in cDCs Is Critical for T Cell Priming.

Inflammasome activation leads to pyroptotic cell death, thereby eliminating the replicative niche of virulent pathogens. Although inflammasome-associated cytokines IL-1ß and IL-18 have an established role in T cell function, whether inflammasome activation in dendritic cells (DCs) is critical for T cell priming is not clear. Here, we find that conventional DCs (cDCs) suppress inflammasome activation to prevent pyroptotic cell death, thus preserving their ability to prime both CD4 and CD8 T cells. Transcription factors IRF8 and IRF4, in cDC1s and cDC2s, respectively, mediate suppression of inflammasome activation by limiting the expression of inflammasome-associated genes. Overexpression of IRF4 or IRF8 inhibits inflammasome activation in macrophages, while reduced expression of IRF8 leads to aberrant inflammasome activation in cDC1s and hampers their ability to prime CD8 T cells. Thus, activation of inflammasome in DCs is detrimental to adaptive immunity, and our results reveal that cDCs use IRF4 and IRF8 to suppress this response.

IRAK1 Is a Critical Mediator of Inflammation-Induced Preterm Birth.

Preterm birth (PTB) is a major cause of neonatal mortality and morbidity, often triggered by chorioamnionitis or intrauterine inflammation (IUI) with or without infection. Recently, there has been a strong association of IL-1 with PTB. We hypothesized that IL-1R-associated kinase 1 (IRAK1), a key signaling mediator in the TLR/IL-1 pathway, plays a critical role in PTB. In human fetal membranes (FM) collected immediately after birth from women delivering preterm, p-IRAK1 was significantly increased in all the layers of FM with chorioamnionitis, compared with no-chorioamnionitis subjects. In a preterm rhesus macaque model of IUI given intra-amniotic LPS, induction of p-IRAK1 and downstream proinflammatory signaling mediators were seen in the FM. In a C57BL/6J wild-type PTB mouse model of IUI given intrauterine LPS, an IRAK1 inhibitor significantly decreased PTB and increased live birth in a dose-dependent manner. Furthermore, IRAK1 knockout mice were protected from LPS-induced PTB, which was seen in wild-type controls. Activation of IRAK1 was maintained by K63-mediated ubiquitination in preterm FM of humans with chorioamnionitis and rhesus and mouse IUI models. Mechanistically, IRAK1 induced PTB in the mouse model of IUI by upregulating expression of COX-2. Thus, our data from human, rhesus, and mouse demonstrates a critical role IRAK1 in IUI and inflammation-associated PTB and suggest it as potential therapeutic target in IUI-induced PTB.

Allergen-Induced C5a/C5aR1 Axis Activation in Pulmonary CD11b+ cDCs Promotes Pulmonary Tolerance through Downregulation of CD40.

Activation of the C5/C5a/C5a receptor 1 (C5aR1) axis during allergen sensitization protects from maladaptive T cell activation. To explore the underlying regulatory mechanisms, we analyzed the impact of C5aR1 activation on pulmonary CD11b+ conventional dendritic cells (cDCs) in the context of house-dust-mite (HDM) exposure. BALB/c mice were intratracheally immunized with an HDM/ovalbumin (OVA) mixture. After 24 h, we detected two CD11b+ cDC populations that could be distinguished on the basis of C5aR1 expression. C5aR1- but not C5aR1+ cDCs strongly induced T cell proliferation of OVA-reactive transgenic CD4+ T cells after re-exposure to antigen in vitro. C5aR1- cDCs expressed higher levels of MHC-II and CD40 than their C5aR1+ counterparts, which correlated directly with a higher frequency of interactions with cognate CD4+ T cells. Priming of OVA-specific T cells by C5aR1+ cDCs could be markedly increased by in vitro blockade of C5aR1 and this was associated with increased CD40 expression. Simultaneous blockade of C5aR1 and CD40L on C5aR1+ cDCs decreased T cell proliferation. Finally, pulsing with OVA-induced C5 production and its cleavage into C5a by both populations of CD11b+ cDCs. Thus, we propose a model in which allergen-induced autocrine C5a generation and subsequent C5aR1 activation in pulmonary CD11b+ cDCs promotes tolerance towards aeroallergens through downregulation of CD40.

Transcriptional profiling identifies caspase-1 as a T cell-intrinsic regulator of Th17 differentiation.

Dendritic cells (DCs) are critical for the differentiation of pathogen-specific CD4 T cells. However, to what extent innate cues from DCs dictate transcriptional changes in T cells remains elusive. Here, we used DCs stimulated with specific pathogens to prime CD4 T cells in vitro and found that these T cells express unique transcriptional profiles dictated by the nature of the priming pathogen. More specifically, the transcriptome of in vitro C. rodentium-primed Th17 cells resembled that of Th17 cells primed following infection in vivo but was remarkably distinct from cytokine-polarized Th17 cells. We identified caspase-1 as a unique gene up-regulated only in pathogen-primed Th17 cells and discovered a critical role for T cell-intrinsic caspase-1, independent of inflammasome, in optimal priming of Th17 responses. T cells lacking caspase-1 failed to induce colitis or confer protection against C. rodentium infection due to suboptimal Th17 cell differentiation in vivo. This study underlines the importance of DC-mediated priming in identifying novel regulators of T cell differentiation.

T cells instruct myeloid cells to produce inflammasome-independent IL-1ß and cause autoimmunity.

The cytokine interleukin (IL)-1ß is a key mediator of antimicrobial immunity as well as autoimmune inflammation. Production of IL-1ß requires transcription by innate immune receptor signaling and maturational cleavage by inflammasomes. Whether this mechanism applies to IL-1ß production seen in T cell-driven autoimmune diseases remains unclear. Here, we describe an inflammasome-independent pathway of IL-1ß production that was triggered upon cognate interactions between effector CD4+ T cells and mononuclear phagocytes (MPs). The cytokine TNF produced by activated CD4+ T cells engaged its receptor TNFR on MPs, leading to pro-IL-1ß synthesis. Membrane-bound FasL, expressed by CD4+ T cells, activated death receptor Fas signaling in MPs, resulting in caspase-8-dependent pro-IL-1ß cleavage. The T cell-instructed IL-1ß resulted in systemic inflammation, whereas absence of TNFR or Fas signaling protected mice from CD4+ T cell-driven autoimmunity. The TNFR-Fas-caspase-8-dependent pathway provides a mechanistic explanation for IL-1ß production and its consequences in CD4+ T cell-driven autoimmune pathology.

An autophagy-inducing and TLR-2 activating BCG vaccine induces a robust protection against tuberculosis in mice.

Mycobacterium bovis BCG is widely used as a vaccine against tuberculosis due to M. tuberculosis (Mtb), which kills millions of people each year. BCG variably protects children, but not adults against tuberculosis. BCG evades phagosome maturation, autophagy, and reduces MHC-II expression of antigen-presenting cells (APCs) affecting T-cell activation. To bypass these defects, an autophagy-inducing, TLR-2 activating C5 peptide from Mtb-derived CFP-10 protein was overexpressed in BCG in combination with Ag85B. Recombinant BCG85C5 induced a robust MHC-II-dependent antigen presentation to CD4 T cells in vitro, and elicited stronger TH1 cytokines (IL-12, IL-1ß, and TNFα) from APCs of C57Bl/6 mice increasing phosphorylation of p38MAPK and ERK. BCG85C5 also enhanced MHC-II surface expression of MΦs by inhibiting MARCH1 ubiquitin ligase that degrades MHC-II. BCG85C5 infected APCs from MyD88 or TLR-2 knockout mice showed decreased antigen presentation. Furthermore, BCG85C5 induced LC3-dependent autophagy in macrophages increasing antigen presentation. Consistent with in vitro effects, BCG85C5 markedly expanded both effector and central memory T cells in C57Bl/6 mice protecting them against both primary aerosol infection with Mtb and reinfection, but was less effective among TLR-2 knockout mice. Thus, BCG85C5 induces stronger and longer lasting immunity, and is better than BCG against tuberculosis of mice.

T cell-intrinsic IL-1R signaling licenses effector cytokine production by memory CD4 T cells.

Innate cytokines are critical drivers of priming and differentiation of naive CD4 T cells, but their functions in memory T cell response are largely undefined. Here we show that IL-1 acts as a licensing signal to permit effector cytokine production by pre-committed Th1 (IFN-γ), Th2 (IL-13, IL-4, and IL-5) and Th17 (IL-17A, IL-17F, and IL-22) lineage cells. This licensing function of IL-1 is conserved across effector CD4 T cells generated by diverse immunological insults. IL-1R signaling stabilizes cytokine transcripts to enable productive and rapid effector functions. We also demonstrate that successful lineage commitment does not translate into productive effector functions in the absence of IL-1R signaling. Acute abrogation of IL-1R signaling in vivo results in reduced IL-17A production by intestinal Th17 cells. These results extend the role of innate cytokines beyond CD4 T cell priming and establish IL-1 as a licensing signal for memory CD4 T cell function.