Jacobsen, Elizabeth A.

Staphylococcus aureus skin colonization and eosinophil infiltration are associated with many inflammatory skin disorders, including atopic dermatitis, bullous pemphigoid, Netherton’s syndrome, and prurigo nodularis. However, whether there is a relationship between S. aureus and eosinophils and how this interaction influences skin inflammation is largely undefined. We show in a preclinical mouse model that S. aureus epicutaneous exposure induced eosinophil-recruiting chemokines and eosinophil infiltration into the skin. Remarkably, we found that eosinophils had a comparable contribution to the skin inflammation as T cells, in a manner dependent on eosinophil-derived IL-17A and IL-17F production. Importantly, IL-36R signaling induced CCL7-mediated eosinophil recruitment to the inflamed skin. Last, S. aureus proteases induced IL-36α expression in keratinocytes, which promoted infiltration of IL-17-producing eosinophils. Collectively, we uncovered a mechanism for S. aureus proteases to trigger eosinophil-mediated skin inflammation, which has implications in the pathogenesis of inflammatory skin diseases.

Abstract Extracellular ATP (eATP) is a danger signal released by inflamed tissues, and can be sensed by immune cells via P2RX7. P2RX7 promotes lung pathology induced by influenza infection, by inducing exacerbated numbers of lung CD4+ T cells. In response to influenza, lung CD4+ T cells are comprised of a type 1 (Th1)-like population, and a subset resembling follicular helper (Tfh) CD4+ T cells; this subset is called tissue-resident helper (Trh). Trh cells amplify lung effector immune responses. Trh cells express high P2RX7 levels, and current research in our laboratory shows that P2RX7 is crucial for Trh cell establishment and subsequent lung damage. We have also found that the intensity of lung damage caused by influenza is positively correlated with P2RX7+ lung Trh cells. It is not clear, however, how concomitant exposure to influenza and type 2 immunity-inducing lung allergens (a common clinical occurrence in the United States) affect P2RX7+ lung Trh cells and their resultant tissue damage. We investigated this, using House Dust Mite (HDM) allergen exposure together with Influenza (PR8) infection. HDM/PR8 co-exposure induced worsening of lung inflammation, compared to single exposure to PR8. This inflammation had type 1 characteristics (i.e. parenchymal immune cell accumulation and absence of mucus). The increased lung damage generated by HDM/PR8 exacerbated the generation of influenza-specific P2RX7+ lung Trh cells. The accumulation of lung Trh cells (and subsequent tissue damage) caused by HDM/PR8 was abolished in T cell-specific P2RX7-knockout mice. Our results suggest that local inflammation, eATP release and sensing, and lung Trh cells together dictate the intensity of lung damage, regardless of the nature of the lung disease agent. R01AI170649

Objective To evaluate eosinophil peroxidase (EPX) as a biomarker for tissue levels of eosinophilia, cytokines, and chemokines within chronic rhinosinusitis (CRS). Methods Twenty‐eight subjects undergoing sinonasal surgery were prospectively enrolled. Ethmoid tissue was analyzed with an in‐house EPX immunoassay and a 48‐plex cytokine‐chemokine array. Clinical severity was assessed using SNOT‐22 and Lund‐Mackay scores. Subjects were grouped as follows: controls, polyp status (CRS with [CRSwNP] and without nasal polyps [CRSsNP]), tissue eosinophilia (eosinophilic CRS [eCRS], non‐eosinophilic CRS [neCRS]), or combinations thereof (eCRSwNP, eCRSsNP, neCRSsNP). eCRS was defined as >10 eosinophils per high power field (HPF). Subjects without CRS or asthma were enrolled as controls. Results EPX was elevated in CRSwNP compared to control ( p = 0.007), in eCRS compared to neCRS ( p = 0.002), and in eCRSwNP along with eCRSsNP compared to neCRSsNP ( p = 0.023, p = 0.015, respectively). eCRS displayed elevated IL‐5 compared to neCRS ( p = 0.005). No significant differences in EPX or IL‐5 were observed between eCRSwNP and eCRSsNP. IL‐5 was elevated in eCRSwNP ( p = 0.019) compared neCRSsNP. Area under the receiver operator characteristic curve was 0.938 (95% CI, 0.835–1.00) for EPX and tissue eosinophilia, with an optimal cut‐point of 470 ng/mL being 100% specific and 81.25% sensitive for tissue eosinophilia. Linear regression revealed a strong correlation between EPX and IL‐5 (R 2 = 0.64, p < 0.001). Comparing EPX and IL‐5, only EPX displayed significant correlation with SNOT‐22 ( p = 0.04) and Lund‐Mackay score ( p = 0.004). Conclusion EPX is associated with tissue eosinophilia in CRS patients regardless of polyp status. EPX correlates with IL‐5 and could be potentially considered a biomarker for anti‐IL‐5 therapies. Level of Evidence 3 Laryngoscope , 134:69–78, 2024

Abstract Eosinophils play a surprising role in downregulating lung graft-specific alloimmunity, even without immunosuppression. We demonstrate here, a mechanism used by eosinophils to downregulate alloimmune responses. To study the mechanism of eosinophil dependent downregulation of alloimmune responses, we used a model of diphtheria toxin (DT) dependent ablation of eosinophils in iPHIL, a mouse model expressing DT receptor on the eosinophil-peroxidase gene locus. Fully MHC-mismatched Balb/c (H2Kd) lungs were engrafted to iPHIL (H2Kb) recipients without immunosuppression. We modeled alloreactivity in vitro, with mixed leukocyte cultures, comprising Th1-polarized eosinophils as modulators of T cells from C57BL/6J or Nur77-GFP mice stimulated by either allogeneic dendritic cells or anti-CD3/CD28 agonistic antibodies. Ablation of eosinophils in non-immunosuppressed lung graft recipients leads to enhanced T cell activation and graft rejection. Similarly, in vitro, we observed a dose and contact dependent downregulation of T cell activation by eosinophils. While contact between eosinophils and T cells is mediated through the upregulation of PD-L1 on both wild-type and iNOS−/− eosinophils after TH-1 polarization, the inhibition of T cell activation is iNOS dependent and is directly linked to the iNOS dependent inhibition of the TCR signaling early gene, Nr4a1, monitored in Nur77-GFP mice. Thus, following contact with T cells, iNOS-expressing eosinophils downregulate lung graft alloimmunity through a direct effect on TCR engagement early genes, leading to the inhibition of T cell activation. This provides an explanation for the surprising observation of lung-graft specific alloimmune downregulation by eosinophils.

Abstract Chronic asthma is characterized by pulmonary remodeling and sustained lung dysfunction. The expression of IL-13 has been demonstrated as sufficient and necessary to induce these pathologies. Despite the common assumption that T cells are the primary source of IL-13 in the lung following allergen provocation, the mechanistic events leading to IL-13 expression are unclear, including the potential importance of other cells (i.e., non-T cells). Methods: Co-expression of IL-5 and eotaxin-2 induces pulmonary histopathologies and lung dysfunction that are eosinophil-dependent (i.e., they are absent in triple transgenic I5/hE2/PHIL) animals. IL-13 knockout and CD4 knockout mice were crossed with I5/hE2 mice to assess the effects of these deficiencies on the induced pathologies occurring in this double transgenic model. Conclusions: The Th2 pulmonary pathologies associated with I5/hE2 mice included recruitment of pulmonary effector T cells and increased brochoalveolar Th2 cytokine levels. Conversely, I5/hE2/CD4-/- had only a ~50% reduction in IL-13 levels as compared to I5/hE2 mice, suggesting that a significant portion of IL-13 is non-T cell derived in these mice. I5/hE2/IL-13-/- mice are improved in lung dysfunction and mucus production as compared to I5/hE2 mice. All mice had similar levels of eosinophil degranulation. These results demonstrate a role for non-T cell derived IL-13 that contributes to mucus production and lung dysfunction in a mouse model of chronic severe asthma.

Abstract Eosinophil infiltration into tumors has been associated with positive and negative growth human cancers and in mouse models of tumorigenesis. Our previous studies have shown that eosinophils infiltrate tumors and differentially accumulate in the necrotic and capsule regions of tumors. Using various eosinophil-deficient mouse models, we demonstrate that in the absence of eosinophils tumor growth is attenuated. Methods: Tumor growth kinetics were assessed using a B16F10 melanoma subcutaneous cell injection model. Eosinophil chemotaxis was assessed using an ex vivo transwell insert assay system. We compared tumor growth in wild type animals vs. various eosinophil-deficient mouse models that are transgenic line of mice (PHIL) and MBP-/-/EPO-/- double knockout mice that are deficient in eosinophil-specific proteins (major basic protein (MBP) and eosinophil peroxide (EPO)) resulting in a blockade of eosinophilpoiesis. Conclusions: The migration of eosinophils was inhibited through blocking the sphingosine-1-phosphate pathway, suggesting recruitment and accumulation is primarily an inflammatory response. Tumor growth was decreased by ~40-60% in PHIL and in MBP-/-/EPO-/- double knockout mice relative to tumors in wild type animals. In addition, EPO-/- mice, and not MBP-/-mice, replicate this ~40% reduction in tumor size implicating a significant role for EPO in tumor growth kinetics. These data suggest eosinophils respond to inflammatory mediators and mediate augment tumor growth.

Abstract Eosinophil effector functions have been hypothesized to be mediated in part by the release of proteins stored in the secondary granules of mature cells of this leukocyte lineage. Major basic protein-1 (MBP-1) and eosinophil peroxidase (EPO) comprise the majority of protein in these granules. No role has been speculated for these proteins in eosinophilopoiesis and/or the accumulation of mature cells in circulation. Methods: Eosinophilopoiesis and eosinophil accumulation in the periphery were assessed in single and double knockout (MBP-1-/- and/or EPO-/-) mice. Bone marrow and peripheral blood leukocytes were assessed by IHC, FACS and in vitro cell culture assays. Mice were subjected to an acute OVA protocol assessing induced pulmonary pathologies in each of the strains of granule protein knockout mice. Results: We demonstrate that, unlike the single deficiency of MBP-1 or EPO, the absence of both granule proteins resulted in the loss of peripheral blood eosinophils. IHC assessments of bone marrow and spleen demonstrate that eosinophil lineage commitment occurs in these mice. However, these assessments and FACS studies demonstrated a blockade in the maturation of eosinophil-lineage committed cells. This blockade is not rescued by ex vivo culture or by bone marrow engraftment into wild type recipient mice. Similar to other eosinophil-less mouse models, MBP-1-/-/EPO-/- mice also fail to develop pulmonary inflammation in an OVA protocol.

Abstract Eosinophil recruitment and accumulation in the lungs of asthmatics is a hallmark feature of asthma. The causative relationship between eosinophils and induced Th2 pulmonary pathologies remains unclear. Our data suggest a novel role for eosinophils as immune modulators that drive Th2-mediated pulmonary pathologies through modulation of dendritic cell function leading to the suppression of Th1/Th17 immune responses. Methods: Eosinophils and ovalbumin (OVA)-pulsed myeloid dendritic cells were adoptively transferred into IL-5-/- (eosinophil-low) or PHIL mice (eosinophil-null) at the time of allergen challenge using an established acute OVA protocol. Allergen-induced pulmonary changes were assessed as changes in lung and lung draining lymph node (LDLN) cells, activation/polarization of T cells, and induced lung histopathologies. Conclusions: We demonstrate that a threshold level of eosinophils must migrate to the lung draining lymph node to promote dendritic cell recruitment and thus activation of antigen-specific memory T cells. Expression of MHC II or CCR7 on eosinophils is not necessary for proliferation of T cells in the LDLN. These lymphatic eosinophils are necessary to suppress Th1/Th17 neutrophilic polarized immune responses initiated by OVA-pulsed dendritic cells during allergen challenge. These results imply an important function for eosinophils in modulating the balance between Th2 and Th1/Th17 pathways in the development of allergic respiratory inflammation.

Background and Aims: A better understanding of the underlying mechanism of acetaminophen (APAP)‐induced liver injury (AILI) remains an important endeavor to develop therapeutic approaches. Eosinophils have been detected in liver biopsies of patients with APAP overdose. We recently demonstrated a profound protective role of eosinophils against AILI; however, the molecular mechanism had not been elucidated. Approach and Results: In agreement with our previous data from experiments using genetic deletion of eosinophils, we found that depletion of eosinophils in wild‐type (WT) mice by an anti‐IL‐15 antibody resulted in exacerbated AILI. Moreover, adoptive transfer of eosinophils significantly reduced liver injury and mortality rate in WT mice. Mechanistic studies using eosinophil‐specific IL‐4/IL‐13 knockout mice demonstrated that these cytokines, through inhibiting interferon‐γ, mediated the hepatoprotective function of eosinophils. Reverse phase protein array analyses and in vitro experiments using various inhibitors demonstrated that IL‐33 stimulation of eosinophils activated p38 mitogen‐activated protein kinase (MAPK), and in turn, cyclooxygenases (COX), which triggered NF‐κB–mediated IL‐4/IL‐13 production. In vivo adoptive transfer experiments showed that in contrast to naive eosinophils, those pretreated with COX inhibitors failed to attenuate AILI. Conclusions: The current study revealed that eosinophil‐derived IL‐4/IL‐13 accounted for the hepatoprotective effect of eosinophils during AILI. The data demonstrated that the p38 MAPK/COX/NF‐κB signaling cascade played a critical role in inducing IL‐4/IL‐13 production by eosinophils in response to IL‐33.

Significance Eosinophils contribute to type 2 immunity against helminths and allergens. The small intestine harbors eosinophils with incompletely understood pathophysiological roles. Here, we show that intestinal eosinophils include two subsets. One expresses the inhibitory receptor Clec4a4 and the inhibitory ligand PD-L1 and is unique to the small intestine; the other manifests a proinflammatory phenotype. Both subsets are blood derived. Remarkably, Clec4a4 + eosinophils were instructed by the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that imprints many gut immune cells. Selective AHR deletion in eosinophils depleted Clec4a4 + eosinophils, augmented innate lymphocytes producing type 2 cytokines, and enhanced helminth clearance. We conclude that Clec4a4 + eosinophils have immunomodulatory functions, which could be harnessed for the therapy of food allergies and eosinophilic gastrointestinal disorders.