Epithelial heme oxygenase-1 enhances colonic tumorigenesis by inhibiting ferroptosis.

Colorectal cancer has been linked to chronic colitis and red meat consumption, which can increase colonic iron and heme. Heme oxygenase-1 ( Hmox1 ) metabolizes heme and releases ferrous iron, but its role in colonic tumorigenesis is not well-described. Recent studies suggest that ferroptosis, the iron-dependent form of cell death, protects against colonic tumorigenesis. Ferroptosis culminates in excessive lipid peroxidation that is constrained by the antioxidative glutathione pathway. We observed increased mucosal markers of ferroptosis and glutathione metabolism in the setting of murine and human colitis, as well as murine colonic neoplasia. We obtained similar results in murine and human colonic epithelial organoids exposed to heme and the ferroptosis activator erastin, especially induction of Hmox1 . RNA sequencing of colonic organoids from mice with deletion of intestinal epithelial Hmox1 (Hmox1 ΔIEC ) revealed increased ferroptosis and activated glutathione metabolism after heme exposure. In a colitis-associated cancer model we observed significantly fewer and smaller tumors in Hmox1 ΔIEC mice compared to littermate controls. Transcriptional profiling of Hmox1 ΔIEC tumors and tumor organoids revealed increased ferroptosis and oxidative stress markers in tumor epithelial cells. In total, our findings reveal ferroptosis as an important colitis-associated cancer signature pathway, and Hmox1 as a key regulator in the tumor microenvironment.

IL-38 regulates intestinal stem cell homeostasis by inducing WNT signaling and beneficial IL-1ß secretion.

The IL-1 Family member IL-38 has been characterized primarily as an antiinflammatory cytokine in human and mouse models of systemic diseases. Here, we examined the role of IL-38 in the murine small intestine (SI). Immunostaining of SI revealed that IL-38 expression partially confines to intestinal stem cells. Cultures of intestinal organoids reveal IL-38 functions as a growth factor by increasing organoid size via inducing WNT3a. In contrast, organoids from IL-38-deficient mice develop more slowly. This reduction in size is likely due to the downregulation of intestinal stemness markers (i.e., Fzd5, Ephb2, and Olfm4) expression compared with wild-type organoids. The IL-38 binding to IL-1R6 and IL-1R9 is still a matter of debate. Therefore, to analyze the molecular mechanisms of IL-38 signaling, we also examined organoids from IL-1R9-deficient mice. Unexpectedly, these organoids, although significantly smaller than wild type, respond to IL-38, suggesting that IL-1R9 is not involved in IL-38 signaling in the stem cell crypt. Nevertheless, silencing of IL-1R6 disabled the organoid response to the growth property of IL-38, thus suggesting IL-1R6 as the main receptor used by IL-38 in the crypt compartment. In organoids from wild-type mice, IL-38 stimulation induced low concentrations of IL-1ß which contribute to organoid growth. However, high concentrations of IL-1ß have detrimental effects on the cultures that were prevented by treatment with recombinant IL-38. Overall, our data demonstrate an important regulatory function of IL-38 as a growth factor, and as an antiinflammatory molecule in the SI, maintaining homeostasis.

Pancreatic Ductal Adenocarcinoma Cells Regulate NLRP3 Activation to Generate a Tolerogenic Microenvironment.

Defining feature of pancreatic ductal adenocarcinoma (PDAC) that participates in the high mortality rate and drug resistance is the immune-tolerant microenvironment which enables tumors to progress unabated by adaptive immunity. In this study, we report that PDAC cells release CSF-1 to induce nucleotide-binding domain, leucine-rich containing family, pyrin domain-containing-3 (NLRP3) activation in myeloid cells. Increased NLRP3 expression was found in the pancreas of patients with PDAC when compared with normal pancreas which correlated with the formation of the NLRP3 inflammasome. Using human primary cells and an orthotopic PDAC mouse model, we show that NLRP3 activation is responsible for the maturation and release of the inflammatory cytokine IL1ß which selectively drives Th2-type inflammation via COX2/PGE2 induction. As a result of this inflammation, primary tumors were characterized by reduced cytotoxic CD8+ T-cell activation and increased tumor expansion. Genetic deletion and pharmacologic inhibition of NLRP3 enabled the development of Th1 immunity, increased intratumoral levels of IL2, CD8+ T cell­mediated tumor suppression, and ultimately limited tumor growth. In addition, we observed that NLRP3 inhibition in combination with gemcitabine significantly increased the efficacy of the chemotherapy. In conclusion, this study provides a mechanism by which tumor-mediated NLRP3 activation exploits a distinct adaptive immunity response that facilitates tumor escape and progression. Considering the ability to block NLRP3 activity with safe and small orally active molecules, this protein represents a new promising target to improve the limited therapeutic options in PDAC. SIGNIFICANT: This study provides novel molecular insights on how PDAC cells exploit NLRP3 activation to suppress CD8 T-cell activation. From a translational perspective, we demonstrate that the combination of gemcitabine with the orally active NLRP3 inhibitor OLT1177 increases the efficacy of monotherapy.

Nucleocapsid protein binding DNA aptamers for detection of SARS-COV-2.

The severe acute respiratory syndrome coronavirus (SARS-CoV-2) has infected millions of individuals and continues to be a major health concern worldwide. While reverse transcription-polymerase chain reaction remains a reliable method for detecting infections, limitations of this technology, particularly cost and the requirement of a dedicated laboratory, prevent rapid viral monitoring. Antigen tests filled this need to some extent but with limitations including sensitivity and specificity, particularly against emerging variants of concern. Here, we developed aptamers against the SARS-CoV-2 Nucleocapsid protein to complement or replace antibodies in antigen detection assays. As detection reagents in ELISA-like assays, our DNA aptamers were able to detect as low as 150 pg/mL of the protein and under 150 k copies of inactivated SARS-CoV-2 Wuhan Alpha strain in viral transport medium with little cross-reactivity to other human coronaviruses (HCoVs). Further, our aptamers were reselected against the SARS-CoV-2 Omicron variant of concern, and we found two sequences that had a more than two-fold increase in signal compared to our original aptamers when used as detection reagents against protein from the Omicron strain. These findings illustrate the use of aptamers as promising alternative detection reagents that may translate for use in current tests and our findings validate the method for the reselection of aptamers against emerging viral strains.

Protective role of tissue-resident Tregs in a murine model of beryllium-induced disease.

CD4+ T cells drive the immunopathogenesis of chronic beryllium disease (CBD), and their recruitment to the lung heralds the onset of granulomatous inflammation. We have shown that CD4+ Tregs control granuloma formation in an HLA-DP2 Tg model of CBD. In these mice, beryllium oxide (BeO) exposure resulted in the accumulation of 3 distinct CD4+ T cell subsets in the lung, with the majority of tissue-resident memory cells expressing FoxP3. The amount of Be regulated the number of total and antigen-specific CD4+ T cells and Tregs in the lungs of HLA-DP2 Tg mice. Depletion of Tregs increased the number of IFN-γ-producing CD4+ T cells and enhanced lung injury, while mice treated with IL-2/αIL-2 complexes had increased Tregs and reduced inflammation and Be-responsive T cells in the lung. BeO-experienced resident Tregs suppressed anti-CD3-induced proliferation of CD4+ T cells in a contact-dependent manner. CTLA-4 and ICOS blockade, as well as the addition of LPS to BeO-exposed mice, increased the effector T cell (Teff)/Treg ratio and enhanced lung injury. Collectively, these data show that the protective role of tissue-resident Tregs is dependent on quantity of Be exposure and is overcome by blocking immune regulatory molecules or additional environmental insults.

Innate and Adaptive Immunity in Noninfectious Granulomatous Lung Disease.

Sarcoidosis and chronic beryllium disease are noninfectious lung diseases that are characterized by the presence of noncaseating granulomatous inflammation. Chronic beryllium disease is caused by occupational exposure to beryllium containing particles, whereas the etiology of sarcoidosis is not known. Genetic susceptibility for both diseases is associated with particular MHC class II alleles, and CD4+ T cells are implicated in their pathogenesis. The innate immune system plays a critical role in the initiation of pathogenic CD4+ T cell responses as well as the transition to active lung disease and disease progression. In this review, we highlight recent insights into Ag recognition in chronic beryllium disease and sarcoidosis. In addition, we discuss the current understanding of the dynamic interactions between the innate and adaptive immune systems and their impact on disease pathogenesis.

Disruption of monocyte-macrophage differentiation and trafficking by a heme analog during active inflammation.

Heme metabolism is a key regulator of inflammatory responses. Cobalt protoporphyrin IX (CoPP) is a heme analog and mimic that potently activates the NRF2/heme oxygenase-1 (HO-1) pathway, especially in monocytes and macrophages. We investigated the influence of CoPP on inflammatory responses using a murine model of colitis. Surprisingly, conditional deletion of myeloid HO-1 did not impact the colonic inflammatory response or the protective influence of CoPP in the setting of dextran sodium sulfate-induced colitis. Rather, we reveal that CoPP elicits a contradictory shift in blood myeloid populations relative to the colon during active intestinal inflammation. Major population changes include markedly diminished trafficking of CCR2+Ly6Chi monocytes to the inflamed colon, despite significant mobilization of this population into circulation. This resulted in significantly diminished colonic expansion of monocyte-derived macrophages and inflammatory cytokine expression. These findings were linked with significant induction of systemic CCL2 leading to a disrupted CCL2 chemoattractant gradient toward the colon and concentration-dependent suppression of circulating monocyte CCR2 expression. Administration of CoPP also induced macrophage differentiation toward a MarcohiHmox1hi anti-inflammatory erythrophagocytic phenotype, contributing to an overall decreased inflammatory profile. Such findings redefine protective influences of heme metabolism during inflammation, and highlight previously unreported immunosuppressive mechanisms of endogenous CCL2 induction.

CD4+ T cells in the lungs of acute sarcoidosis patients recognize an Aspergillus nidulans epitope.

Löfgren's syndrome (LS) is an acute form of sarcoidosis characterized by a genetic association with HLA-DRB1*03 (HLA-DR3) and an accumulation of CD4+ T cells of unknown specificity in the bronchoalveolar lavage (BAL). Here, we screened related LS-specific TCRs for antigen specificity and identified a peptide derived from NAD-dependent histone deacetylase hst4 (NDPD) of Aspergillus nidulans that stimulated these CD4+ T cells in an HLA-DR3-restricted manner. Using ELISPOT analysis, a greater number of IFN-γ- and IL-2-secreting T cells in the BAL of DR3+ LS subjects compared with DR3+ control subjects was observed in response to the NDPD peptide. Finally, increased IgG antibody responses to A. nidulans NDPD were detected in the serum of DR3+ LS subjects. Thus, our findings identify a ligand for CD4+ T cells derived from the lungs of LS patients and suggest a role of A. nidulans in the etiology of LS.

Beryllium-specific CD4+ T cells induced by chemokine neoantigens perpetuate inflammation.

Discovering dominant epitopes for T cells, particularly CD4+ T cells, in human immune-mediated diseases remains a significant challenge. Here, we used bronchoalveolar lavage (BAL) cells from HLA-DP2-expressing patients with chronic beryllium disease (CBD), a debilitating granulomatous lung disorder characterized by accumulations of beryllium-specific (Be-specific) CD4+ T cells in the lung. We discovered lung-resident CD4+ T cells that expressed a disease-specific public CDR3ß T cell receptor motif and were specific to Be-modified self-peptides derived from C-C motif ligand 4 (CCL4) and CCL3. HLA-DP2-CCL/Be tetramer staining confirmed that these chemokine-derived peptides represented major antigenic targets in CBD. Furthermore, Be induced CCL3 and CCL4 secretion in the lungs of mice and humans. In a murine model of CBD, the addition of LPS to Be oxide exposure enhanced CCL4 and CCL3 secretion in the lung and significantly increased the number and percentage of CD4+ T cells specific for the HLA-DP2-CCL/Be epitope. Thus, we demonstrate a direct link between Be-induced innate production of chemokines and the development of a robust adaptive immune response to those same chemokines presented as Be-modified self-peptides, creating a cycle of innate and adaptive immune activation.

Type 1 interferon-dependent repression of NLRC4 and iPLA2 licenses down-regulation of Salmonella flagellin inside macrophages.

Inflammasomes have been implicated in the detection and clearance of a variety of bacterial pathogens, but little is known about whether this innate sensing mechanism has any regulatory effect on the expression of stimulatory ligands by the pathogen. During infection with Salmonella and many other pathogens, flagellin is a major activator of NLRC4 inflammasome-mediated macrophage pyroptosis and pathogen eradication. Salmonella switches to a flagellin-low phenotype as infection progresses to avoid this mechanism of clearance by the host. However, the host cues that Salmonella perceives to undergo this switch remain unclear. Here, we report an unexpected role of the NLRC4 inflammasome in promoting expression of its microbial ligand, flagellin, and identify a role for type 1 IFN signaling in switching of Salmonella to a flagellin-low phenotype. Early in infection, activation of NLRC4 by flagellin initiates pyroptosis and concomitant release of lysophospholipids which in turn enhance expression of flagellin by Salmonella thereby amplifying its ability to elicit cell death. TRIF-dependent production of type 1 IFN, however, later represses NLRC4 and the lysophospholipid biosynthetic enzyme iPLA2, causing a decline in intracellular lysophospholipids that results in down-regulation of flagellin expression by Salmonella These findings reveal a previously unrecognized immune-modulating regulatory cross-talk between endosomal TLR signaling and cytosolic NLR activation with significant implications for the establishment of infection with Salmonella.

Human and Mouse Transcriptome Profiling Identifies Cross-Species Homology in Pulmonary and Lymph Node Mononuclear Phagocytes.

The mononuclear phagocyte (MP) system consists of macrophages, monocytes, and dendritic cells (DCs). MP subtypes play distinct functional roles in steady-state and inflammatory conditions. Although murine MPs are well characterized, their pulmonary and lymph node (LN) human homologs remain poorly understood. To address this gap, we have created a gene expression compendium across 24 distinct human and murine lung and LN MPs, along with human blood and murine spleen MPs, to serve as validation datasets. In-depth RNA sequencing identifies corresponding human-mouse MP subtypes and determines marker genes shared and divergent across species. Unexpectedly, only 13%-23% of the top 1,000 marker genes (i.e., genes not shared across species-specific MP subtypes) overlap in corresponding human-mouse MP counterparts. Lastly, CD88 in both species helps distinguish monocytes/macrophages from DCs. Our cross-species expression compendium serves as a resource for future translational studies to investigate beforehand whether pursuing specific MP subtypes or genes will prove fruitful.

HIV Infection Is Associated with Loss of Anti-Inflammatory Alveolar Macrophages.

HIV type 1 is associated with pulmonary dysfunction that is exacerbated by cigarette smoke. Alveolar macrophages (AM) are the most prominent immune cell in the alveolar space. These cells play an important role in clearing inhaled pathogens and regulating the inflammatory environment; however, how HIV infection impacts AM phenotype and function is not well understood, in part because of their autofluorescence and the absence of well-defined surface markers. The main aim of this study was to evaluate the impact of HIV infection on human AM and to compare the effect of smoking on their phenotype and function. Time-of-flight mass cytometry and RNA sequencing were used to characterize macrophages from human bronchoalveolar lavage of HIV-infected and -uninfected smokers and nonsmokers. We found that the frequency of CD163+ anti-inflammatory AM was decreased, whereas CD163-CCR7+ proinflammatory AM were increased in HIV infection. HIV-mediated proinflammatory polarization was associated with increased levels of inflammatory cytokines and macrophage activation. Conversely, smoking heightened the inflammatory response evident by change in the expression of CXCR4 and TLR4. Altogether, these findings suggest that HIV infection, along with cigarette smoke, favors a proinflammatory macrophage phenotype associated with enhanced expression of inflammatory molecules. Further, this study highlights time-of-flight mass cytometry as a reliable method for immunophenotyping the highly autofluorescent cells present in the bronchoalveolar lavage of cigarette smokers.

A role for TNF-α in alveolar macrophage damage-associated molecular pattern release.

Chronic beryllium disease (CBD) is a metal hypersensitivity/autoimmune disease in which damage-associated molecular patterns (DAMPs) promote a break in T cell tolerance and expansion of Be2+/self-peptide-reactive CD4+ T cells. In this study, we investigated the mechanism of cell death induced by beryllium particles in alveolar macrophages (AMs) and its impact on DAMP release. We found that phagocytosis of Be led to AM cell death independent of caspase, receptor-interacting protein kinases 1 and 3, or ROS activity. Before cell death, Be-exposed AMs secreted TNF-α that boosted intracellular stores of IL-1α followed by caspase-8-dependent fragmentation of DNA. IL-1α and nucleosomal DNA were subsequently released from AMs upon loss of plasma membrane integrity. In contrast, necrotic AMs released only unfragmented DNA and necroptotic AMs released only IL-1α. In mice exposed to Be, TNF-α promoted release of DAMPs and was required for the mobilization of immunogenic DCs, the expansion of Be-reactive CD4+ T cells, and pulmonary inflammation in a mouse model of CBD. Thus, early autocrine effects of particle-induced TNF-α on AMs led to a break in peripheral tolerance. This potentially novel mechanism may underlie the known relationship between fine particle inhalation, TNF-α, and loss of peripheral tolerance in T cell-mediated autoimmune disease and hypersensitivities.

Adaptive Immunity in Pulmonary Sarcoidosis and Chronic Beryllium Disease.

Pulmonary sarcoidosis and chronic beryllium disease (CBD) are inflammatory granulomatous lung diseases defined by the presence of non-caseating granulomas in the lung. CBD results from beryllium exposure in the workplace, while the cause of sarcoidosis remains unknown. CBD and sarcoidosis are both immune-mediated diseases that involve Th1-polarized inflammation in the lung. Beryllium exposure induces trafficking of dendritic cells to the lung in a mechanism dependent on MyD88 and IL-1α. B cells are also recruited to the lung in a MyD88 dependent manner after beryllium exposure in order to protect the lung from beryllium-induced injury. Similar to most immune-mediated diseases, disease susceptibility in CBD and sarcoidosis is driven by the expression of certain MHCII molecules, primarily HLA-DPB1 in CBD and several HLA-DRB1 alleles in sarcoidosis. One of the defining features of both CBD and sarcoidosis is an infiltration of activated CD4+ T cells in the lung. CD4+ T cells in the bronchoalveolar lavage (BAL) of CBD and sarcoidosis patients are highly Th1 polarized, and there is a significant increase in inflammatory Th1 cytokines present in the BAL fluid. In sarcoidosis, there is also a significant population of Th17 cells in the lungs that is not present in CBD. Due to persistent antigen exposure and chronic inflammation in the lung, these activated CD4+ T cells often display either an exhausted or anergic phenotype. Evidence suggests that these T cells are responding to common antigens in the lung. In CBD there is an expansion of beryllium-responsive TRBV5.1+ TCRs expressed on pathogenic CD4+ T cells derived from the BAL of CBD patients that react with endogenous human peptides derived from the plexin A protein. In an acute form of sarcoidosis, there are expansions of specific TRAV12-1/TRBV2 T cell receptors expressed on BAL CD4+ T cells, indicating that these T cells are trafficking to and expanding in the lung in response to common antigens. The specificity of these pathogenic CD4+T cells in sarcoidosis are currently unknown.

Protective role of B cells in sterile particulate-induced lung injury.

Susceptibility to chronic beryllium (Be) disease is linked to HLA-DP molecules possessing a glutamic acid at the 69th position of the ß-chain (ßGlu69), with the most prevalent ßGlu69-containing molecule being HLA-DP2. We have previously shown that HLA-DP2 transgenic (Tg) mice exposed to Be oxide (BeO) develop mononuclear infiltrates in a peribronchovascular distribution and a beryllium-specific, HLA-DP2-restricted CD4+ T cell response. In addition to T cells, B cells constituted a major portion of infiltrated leukocytes in the lung of BeO-exposed HLA-DP2 Tg mice and sequester BeO particles within ectopic lymphoid aggregates and granulomas. B cell depletion was associated with a loss of lymphoid aggregates and granulomas as well as a significant increase in lung injury in BeO-exposed mice. The protective role of B cells was innate in origin, and BeO-induced B cell recruitment to the lung was dependent on MyD88 signaling. Similar to BeO-exposed HLA-DP2 mice, B cells also accumulate in the lungs of CBD subjects, located at the periphery and surrounding the granuloma. Overall, our data suggest a novel modulatory role for B cells in the protection of the lung against sterile particulate exposure, with B cell recruitment to the inflamed lung occurring in an antigen-independent and MyD88-dependent manner.

Immune Surveillance by Natural IgM Is Required for Early Neoantigen Recognition and Initiation of Adaptive Immunity.

Early recognition of neoantigen-expressing cells is complex, involving multiple immune cell types. In this study, in vivo, we examined how antigen-presenting cell subtypes coordinate and induce an immunological response against neoantigen-expressing cells, particularly in the absence of a pathogen-associated molecular pattern, which is normally required to license antigen-presenting cells to present foreign or self-antigens as immunogens. Using two reductionist models of neoantigen-expressing cells and two cancer models, we demonstrated that natural IgM is essential for the recognition and initiation of adaptive immunity against neoantigen-expressing cells. Natural IgM antibodies form a cellular immune complex with the neoantigen-expressing cells. This immune complex licenses surveying monocytes to present neoantigens as immunogens to CD4+ T cells. CD4+ T helper cells, in turn, use CD40L to license cross-presenting CD40+ Batf3+ dendritic cells to elicit a cytotoxic T cell response against neoantigen-expressing cells. Any break along this immunological chain reaction results in the escape of neoantigen-expressing cells. This study demonstrates the surprising, essential role of natural IgM as the initiator of a sequential signaling cascade involving multiple immune cell subtypes. This sequence is required to coordinate an adaptive immune response against neoantigen-expressing cells.

Isolation and Identification of Interstitial Macrophages from the Lungs Using Different Digestion Enzymes and Staining Strategies.

Interstitial macrophages (IMs) are present in multiple organs. Although there is limited knowledge of the unique functional role IM subtypes play, macrophages, in general, are known for their contribution in homeostatic tissue maintenance and inflammation such as clearing pathogens and debris and secreting inflammatory mediators and growth factors. IM subtypes have been identified in the heart, skin, and gut, and more recently we identified three distinct IMs in the lung. IMs express on their surface high levels of MerTK, CD64, and CD11b, with differences in CD11c, CD206, and MHC II expression, and referred to the three pulmonary IM subtypes as IM1 (CD11cloCD206+MHCIIlo), IM2 (CD11cloCD206+MHCIIhi), and IM3 (CD11chiCD206loMHCIIhi). In this chapter, we highlight how to extract IMs from the lung using three different digestion enzymes: elastase, collagenase D, and Liberase TM. Of these three commonly used enzymes, Liberase TM was the most effective at IM extraction, particularly IM3. Furthermore, alternative staining strategies to identify IMs were examined, which included CD64, MerTK, F4/80, and Tim4. Thus, future studies highlighting the functional role of IM subtypes will help further our understanding of how tissue homeostasis is maintained and inflammatory conditions are induced and resolved.

Three Unique Interstitial Macrophages in the Murine Lung at Steady State.

The current paradigm in macrophage biology is that some tissues mainly contain macrophages from embryonic origin, such as microglia in the brain, whereas other tissues contain postnatal-derived macrophages, such as the gut. However, in the lung and in other organs, such as the skin, there are both embryonic and postnatal-derived macrophages. In this study, we demonstrate in the steady-state lung that the mononuclear phagocyte system is comprised of three newly identified interstitial macrophages (IMs), alveolar macrophages, dendritic cells, and few extravascular monocytes. We focused on similarities and differences between the three IM subtypes, specifically, their phenotype, location, transcriptional signature, phagocytic capacity, turnover, and lack of survival dependency on fractalkine receptor, CX3CR1. Pulmonary IMs were located in the bronchial interstitium but not the alveolar interstitium. At the transcriptional level, all three IMs displayed a macrophage signature and phenotype. All IMs expressed MER proto-oncogene, tyrosine kinase, CD64, CD11b, and CX3CR1, and were further distinguished by differences in cell surface protein expression of CD206, Lyve-1, CD11c, CCR2, and MHC class II, along with the absence of Ly6C, Ly6G, and Siglec F. Most intriguingly, in addition to the lung, similar phenotypic populations of IMs were observed in other nonlymphoid organs, perhaps highlighting conserved functions throughout the body. These findings promote future research to track four distinct pulmonary macrophages and decipher the division of labor that exists between them.