Kidney resident macrophages have distinct subsets and multifunctional roles.

BACKGROUND: The kidney contains distinct glomerular and tubulointerstitial compartments with diverse cell types and extracellular matrix components. The role of immune cells in glomerular environment is crucial for dampening inflammation and maintaining homeostasis. Macrophages are innate immune cells that are influenced by their tissue microenvironment. However, the multifunctional role of kidney macrophages remains unclear. METHODS: Flow and imaging cytometry were used to determine the relative expression of CD81 and CX3CR1 (C-X3-C motif chemokine receptor 1) in kidney macrophages. Monocyte replenishment was assessed in Cx3cr1CreER X R26-yfp-reporter and shielded chimeric mice. Bulk RNA-sequencing and mass spectrometry-based proteomics were performed on isolated kidney macrophages from wild type and Col4a5-/- (Alport) mice. RNAscope was used to visualize transcripts and macrophage purity in bulk RNA assessed by CIBERSORTx analyses. RESULTS: In wild type mice we identified three distinct kidney macrophage subsets using CD81 and CX3CR1 and these subsets showed dependence on monocyte replenishment. In addition to their immune function, bulk RNA-sequencing of macrophages showed enrichment of biological processes associated with extracellular matrix. Proteomics identified collagen IV and laminins in kidney macrophages from wild type mice whilst other extracellular matrix proteins including cathepsins, ANXA2 and LAMP2 were enriched in Col4a5-/- (Alport) mice. A subset of kidney macrophages co-expressed matrix and macrophage transcripts. CONCLUSIONS: We identified CD81 and CX3CR1 positive kidney macrophage subsets with distinct dependence for monocyte replenishment. Multiomic analysis demonstrated that these cells have diverse functions that underscore the importance of macrophages in kidney health and disease.

T helper 2 cells control monocyte to tissue-resident macrophage differentiation during nematode infection of the pleural cavity.

The recent revolution in tissue-resident macrophage biology has resulted largely from murine studies performed in C57BL/6 mice. Here, using both C57BL/6 and BALB/c mice, we analyze immune cells in the pleural cavity. Unlike C57BL/6 mice, naive tissue-resident large-cavity macrophages (LCMs) of BALB/c mice failed to fully implement the tissue-residency program. Following infection with a pleural-dwelling nematode, these pre-existing differences were accentuated with LCM expansion occurring in C57BL/6, but not in BALB/c mice. While infection drove monocyte recruitment in both strains, only in C57BL/6 mice were monocytes able to efficiently integrate into the resident pool. Monocyte-to-macrophage conversion required both T cells and interleukin-4 receptor alpha (IL-4Rα) signaling. The transition to tissue residency altered macrophage function, and GATA6+ tissue-resident macrophages were required for host resistance to nematode infection. Therefore, during tissue nematode infection, T helper 2 (Th2) cells control the differentiation pathway of resident macrophages, which determines infection outcome.

Monocyte migration profiles define disease severity in acute COVID-19 and unique features of long COVID.

BACKGROUND: COVID-19 is associated with a dysregulated immune response but it is unclear how immune dysfunction contributes to the chronic morbidity persisting in many COVID-19 patients during convalescence (long COVID). METHODS: We assessed phenotypical and functional changes of monocytes in COVID-19 patients during hospitalisation and up to 9 months of convalescence following COVID-19, respiratory syncytial virus or influenza A. Patients with progressive fibrosing interstitial lung disease were included as a positive control for severe, ongoing lung injury. RESULTS: Monocyte alterations in acute COVID-19 patients included aberrant expression of leukocyte migration molecules, continuing into convalescence (n=142) and corresponding with specific symptoms of long COVID. Long COVID patients with unresolved lung injury, indicated by sustained shortness of breath and abnormal chest radiology, were defined by high monocyte expression of C-X-C motif chemokine receptor 6 (CXCR6) (p<0.0001) and adhesion molecule P-selectin glycoprotein ligand 1 (p<0.01), alongside preferential migration of monocytes towards the CXCR6 ligand C-X-C motif chemokine ligand 16 (CXCL16) (p<0.05), which is abundantly expressed in the lung. Monocyte CXCR6 and lung CXCL16 were heightened in patients with progressive fibrosing interstitial lung disease (p<0.001), confirming a role for the CXCR6-CXCL16 axis in ongoing lung injury. Conversely, monocytes from long COVID patients with ongoing fatigue exhibited a sustained reduction of the prostaglandin-generating enzyme cyclooxygenase 2 (p<0.01) and CXCR2 expression (p<0.05). These monocyte changes were not present in respiratory syncytial virus or influenza A convalescence. CONCLUSIONS: Our data define unique monocyte signatures that define subgroups of long COVID patients, indicating a key role for monocyte migration in COVID-19 pathophysiology. Targeting these pathways may provide novel therapeutic opportunities in COVID-19 patients with persistent morbidity.

Consequences of collagen induced inflammatory arthritis on circadian regulation of the gut microbiome.

The gut microbiota is important for host health and immune system function. Moreover autoimmune diseases, such as rheumatoid arthritis, are associated with significant gut microbiota dysbiosis, although the causes and consequences of this are not fully understood. It has become clear that the composition and metabolic outputs of the microbiome exhibit robust 24 h oscillations, a result of daily variation in timing of food intake as well as rhythmic circadian clock function in the gut. Here, we report that experimental inflammatory arthritis leads to a re-organization of circadian rhythmicity in both the gut and associated microbiome. Mice with collagen induced arthritis exhibited extensive changes in rhythmic gene expression in the colon, and reduced barrier integrity. Re-modeling of the host gut circadian transcriptome was accompanied by significant alteration of the microbiota, including widespread loss of rhythmicity in symbiont species of Lactobacillus, and alteration in circulating microbial derived factors, such as tryptophan metabolites, which are associated with maintenance of barrier function and immune cell populations within the gut. These findings highlight that altered circadian rhythmicity during inflammatory disease contributes to dysregulation of gut integrity and microbiome function.

The wild mouse bone marrow has a unique myeloid and lymphoid composition and phenotype.

The murine bone marrow has a central role in immune function and health as the primary source of leukocytes in adult mice. Laboratory mice provide a human-homologous, genetically manipulable and reproducible model that has enabled an immeasurable volume of high-quality immunological research. However, recent research has questioned the translatability of laboratory mouse research into humans and proposed that the exposure of mice to their wild and natural environment may hold the key to further immunological breakthroughs. To date, there have been no studies providing an in-depth cellular analysis of the wild mouse bone marrow. This study utilized wild mice from an isolated island population (Isle of May, Scotland, UK) and performed flow cytometric and histological analysis to characterize the myeloid, lymphoid, hematopoietic progenitor, and adipocyte compartments within the wild mouse bone marrow. We find that, compared to laboratory mouse bone marrow, the wild mouse bone marrow differs in every cell type assessed. Some of the major distinctions include; a smaller B cell compartment with an enriched presence of plasma cells, increased proportions of KLRG1+ CD8+ T cells, diminished CD11b expression in the myeloid lineage and a five-fold enlargement of the eosinophil compartment. We conclude that the wild mouse bone marrow is dramatically distinct from its laboratory counterparts, with multiple phenotypes that to our knowledge have never been observed in laboratory models. Further research into these unique features may uncover novel immunological mechanisms and grant a greater understanding of the role of the immune system in a natural setting.

BMPR1a Is Required for the Optimal TGFß1-Dependent CD207+ Langerhans Cell Differentiation and Limits Skin Inflammation through CD11c+ Cells.

The cytokine TGFß1 induces epidermal Langerhans cell (LC) differentiation from human precursors, an effect mediated through BMPR1a/ALK3 signaling, as revealed from ectopic expression and receptor inhibition studies. Whether TGFß1‒BMPR1a signaling is required for LC differentiation in vivo remained incompletely understood. We found that TGFß1-deficient mice show defective perinatal expansion and differentiation of LCs. LCs can be identified within the normal healthy human epidermis by anti-BMPR1a immunohistology staining. Deletion of BMPR1a in all (vav+) hematopoietic cells revealed that BMPR1a is required for the efficient TGFß1-dependent generation of CD207+ LC-like cells from CD11c+ intermediates in vitro. Similarly, BMPR1a was required for the optimal induction of CD207 by preformed major histocompatibility complex II‒positive epidermal resident LC precursors in the steady state. BMPR1a expression is strongly upregulated in epidermal cells in psoriatic lesions, and BMPR1aΔCD11c mice showed a defect in the resolution phase of allergic and psoriatic skin inflammation. Moreover, whereas LCs from these mice expressed CD207, BMPR1a counteracted LC activation and migration from skin explant cultures. Therefore, TGFß1‒BMPR1a signaling seems to be required for the efficient induction of CD207 during LC differentiation in the steady state, and bone marrow‒derived lesional CD11c+ cells may limit established skin inflammation through enhanced BMPR1a signaling.

Gingival monocytes: Lessons from other barriers.

Unlike other non-lymphoid tissues monocytes comprise a large proportion of mononuclear phagocytes present within the gingiva. Their functions and fate remain poorly understood. The oral mucosa faces challenges common to all barrier surfaces, including constant exposure to antigens and the resident commensal bacteria, but also experiences ongoing mechanical damage from mastication. Gingiva monocytes may therefore possess both myeloid functions observed at other barrier sites, such as hypo-responsiveness to bacterial stimulation, and distinctive functions tailored by their unique environment. In this review, we discuss the establishment and function of monocytes and macrophages at several mucosal tissues, and posit potential functions of monocytes within the gingiva tissue.

Integrated miRNA/cytokine/chemokine profiling reveals severity-associated step changes and principal correlates of fatality in COVID-19.

Inflammatory cytokines and chemokines (CC) drive COVID-19 pathology. Yet, patients with similar circulating CC levels present with different disease severity. Here, we determined 171 microRNAomes from 58 hospitalized COVID-19 patients (Cohort 1) and levels of 25 cytokines and chemokines (CC) in the same samples. Combining microRNA (miRNA) and CC measurements allowed for discrimination of severe cases with greater accuracy than using miRNA or CC levels alone. Severity group-specific associations between miRNAs and COVID-19-associated CC (e.g., IL6, CCL20) or clinical hallmarks of COVID-19 (e.g., neutrophilia, hypoalbuminemia) separated patients with similar CC levels but different disease severity. Analysis of an independent cohort of 108 patients from a different center (Cohort 2) demonstrated feasibility of CC/miRNA profiling in leftover hospital blood samples with similar severe disease CC and miRNA profiles, and revealed CCL20, IL6, IL10, and miR-451a as key correlates of fatal COVID-19. These findings highlight that systemic miRNA/CC networks underpin severe COVID-19.

Alterations in T and B cell function persist in convalescent COVID-19 patients.

BACKGROUND: Emerging studies indicate that some coronavirus disease 2019 (COVID-19) patients suffer from persistent symptoms, including breathlessness and chronic fatigue; however, the long-term immune response in these patients presently remains ill-defined. METHODS: Here, we describe the phenotypic and functional characteristics of B and T cells in hospitalized COVID-19 patients during acute disease and at 3-6 months of convalescence. FINDINGS: We report that the alterations in B cell subsets observed in acute COVID-19 patients were largely recovered in convalescent patients. In contrast, T cells from convalescent patients displayed continued alterations with persistence of a cytotoxic program evident in CD8+ T cells as well as elevated production of type 1 cytokines and interleukin-17 (IL-17). Interestingly, B cells from patients with acute COVID-19 displayed an IL-6/IL-10 cytokine imbalance in response to Toll-like receptor activation, skewed toward a pro-inflammatory phenotype. Whereas the frequency of IL-6+ B cells was restored in convalescent patients irrespective of clinical outcome, the recovery of IL-10+ B cells was associated with the resolution of lung pathology. CONCLUSIONS: Our data detail lymphocyte alterations in previously hospitalized COVID-19 patients up to 6 months following hospital discharge and identify 3 subgroups of convalescent patients based on distinct lymphocyte phenotypes, with 1 subgroup associated with poorer clinical outcome. We propose that alterations in B and T cell function following hospitalization with COVID-19 could affect longer-term immunity and contribute to some persistent symptoms observed in convalescent COVID-19 patients. FUNDING: Provided by UKRI, Lister Institute of Preventative Medicine, the Wellcome Trust, The Kennedy Trust for Rheumatology Research, and 3M Global Giving.

Hematopoietic stem and progenitor cells are present in healthy gingiva tissue.

Hematopoietic stem cells reside in the bone marrow, where they generate the effector cells that drive immune responses. However, in response to inflammation, some hematopoietic stem and progenitor cells (HSPCs) are recruited to tissue sites and undergo extramedullary hematopoiesis. Contrasting with this paradigm, here we show residence and differentiation of HSPCs in healthy gingiva, a key oral barrier in the absence of overt inflammation. We initially defined a population of gingiva monocytes that could be locally maintained; we subsequently identified not only monocyte progenitors but also diverse HSPCs within the gingiva that could give rise to multiple myeloid lineages. Gingiva HSPCs possessed similar differentiation potentials, reconstitution capabilities, and heterogeneity to bone marrow HSPCs. However, gingival HSPCs responded differently to inflammatory insults, responding to oral but not systemic inflammation. Combined, we highlight a novel pathway of myeloid cell development at a healthy barrier, defining a gingiva-specific HSPC network that supports generation of a proportion of the innate immune cells that police this barrier.

The Helminth Parasite Heligmosomoides polygyrus Attenuates EAE in an IL-4Rα-Dependent Manner.

Helminth parasites are effective in biasing Th2 immunity and inducing regulatory pathways that minimize excessive inflammation within their hosts, thus allowing chronic infection to occur whilst also suppressing bystander atopic or autoimmune diseases. Multiple sclerosis (MS) is a severe autoimmune disease characterized by inflammatory lesions within the central nervous system; there are very limited therapeutic options for the progressive forms of the disease and none are curative. Here, we used the experimental autoimmune encephalomyelitis (EAE) model to examine if the intestinal helminth Heligmosomoides polygyrus and its excretory/secretory products (HES) are able to suppress inflammatory disease. Mice infected with H. polygyrus at the time of immunization with the peptide used to induce EAE (myelin-oligodendrocyte glycoprotein, pMOG), showed a delay in the onset and peak severity of EAE disease, however, treatment with HES only showed a marginal delay in disease onset. Mice that received H. polygyrus 4 weeks prior to EAE induction were also not significantly protected. H. polygyrus secretes a known TGF-ß mimic (Hp-TGM) and simultaneous H. polygyrus infection with pMOG immunization led to a significant expansion of Tregs; however, administering the recombinant Hp-TGM to EAE mice failed to replicate the EAE protection seen during infection, indicating that this may not be central to the disease protecting mechanism. Mice infected with H. polygyrus also showed a systemic Th2 biasing, and restimulating splenocytes with pMOG showed release of pMOG-specific IL-4 as well as suppression of inflammatory IL-17A. Notably, a Th2-skewed response was found only in mice infected with H. polygyrus at the time of EAE induction and not those with a chronic infection. Furthermore, H. polygyrus failed to protect against disease in IL-4Rα-/- mice. Together these results indicate that the EAE disease protective mechanism of H. polygyrus is likely to be predominantly Th2 deviation, and further highlights Th2-biasing as a future therapeutic strategy for MS.

Longitudinal immune profiling reveals key myeloid signatures associated with COVID-19.

COVID-19 pathogenesis is associated with an exaggerated immune response. However, the specific cellular mediators and inflammatory components driving diverse clinical disease outcomes remain poorly understood. We undertook longitudinal immune profiling on both whole blood and peripheral blood mononuclear cells (PBMCs) of hospitalized patients during the peak of the COVID-19 pandemic in the UK. Here, we report key immune signatures present shortly after hospital admission that were associated with the severity of COVID-19. Immune signatures were related to shifts in neutrophil to T cell ratio, elevated serum IL-6, MCP-1 and IP-10, and most strikingly, modulation of CD14+ monocyte phenotype and function. Modified features of CD14+ monocytes included poor induction of the prostaglandin-producing enzyme, COX-2, as well as enhanced expression of the cell cycle marker Ki-67. Longitudinal analysis revealed reversion of some immune features back to the healthy median level in patients with a good eventual outcome. These findings identify previously unappreciated alterations in the innate immune compartment of COVID-19 patients and lend support to the idea that therapeutic strategies targeting release of myeloid cells from bone marrow should be considered in this disease. Moreover, they demonstrate that features of an exaggerated immune response are present early after hospital admission suggesting immune-modulating therapies would be most beneficial at early timepoints.

Ligature-induced periodontitis induces systemic inflammation but does not alter acute outcome after stroke in mice.

BACKGROUND: Stroke is a major cause of disability and mortality. Poorer outcome after stroke is associated with concomitant inflammatory and infectious disease. Periodontitis is a chronic inflammatory disease of the dental supporting structures and is a prominent risk factor for many systemic disorders, including cardiovascular disease and stroke. While epidemiological studies suggest that periodontitis increases the likelihood of stroke, its impact on stroke severity is poorly understood. Here, we sought to determine the contribution of periodontitis to acute stroke pathology. METHODS: We characterized a murine ligature model of periodontitis for inflammatory responses that could potentially impact stroke outcome. We applied this model and then subjected mice to either transient or permanent middle cerebral artery occlusion. We also enhanced the periodontitis model with repeated intravenous administration of a periodontal-specific lipopolysaccharide to better mimic the clinical condition. RESULTS: Ligature-induced periodontitis caused bone loss, bacterial growth, and increased local inflammatory cell trafficking. Systemically, periodontitis increased circulating levels of pro-inflammatory cytokines, and primed bone marrow monocytes to produce elevated tumour necrosis factor-alpha (TNFα). Despite these changes, periodontitis alone or in tandem with repeated lipopolysaccharide challenge did not alter infarct volume, blood-brain barrier breakdown, or systemic inflammation after experimental stroke. CONCLUSIONS: Our data show that despite elevated systemic inflammation in periodontitis, oral inflammatory disease does not impact acute stroke pathology in terms of severity, determined primarily by infarct volume. This indicates that, at least in this experimental paradigm, periodontitis alone does not alter acute outcome after cerebral ischemia.

Does the Microbiome Affect the Outcome of Renal Transplantation?

The role of the human microbiome in health and disease is becoming increasingly apparent. Emerging evidence suggests that the microbiome is affected by solid organ transplantation. Kidney transplantation is the gold standard treatment for End-Stage Renal Disease (ESRD), the advanced stage of Chronic Kidney Disease (CKD). The question of how ESRD and transplantation affect the microbiome and vice versa includes how the microbiome is affected by increased concentrations of toxins such as urea and creatinine (which are elevated in ESRD), whether restoration of renal function following transplantation alters the composition of the microbiome, and the impact of lifelong administration of immunosuppressive drugs on the microbiome. Changes in microbiome composition and activity have been reported in ESRD and in therapeutic immunosuppression, but the effect on the outcome of transplantation is not well-understood. Here, we consider the current evidence that changes in kidney function and immunosuppression following transplantation influence the oral, gut, and urinary microbiomes in kidney transplant patients. The potential for changes in these microbiomes to lead to disease, systemic inflammation, or rejection of the organ itself is discussed, along with the possibility that restoration of kidney function might re-establish orthobiosis.

Distal Consequences of Oral Inflammation.

Periodontitis is an incredibly prevalent chronic inflammatory disease, which results in the destruction of tooth supporting structures. However, in addition to causing tooth and alveolar bone loss, this oral inflammatory disease has been shown to contribute to disease states and inflammatory pathology at sites distant from the oral cavity. Epidemiological and experimental studies have linked periodontitis to the development and/or exacerbation of a plethora of other chronic diseases ranging from rheumatoid arthritis to Alzheimer's disease. Such studies highlight how the inflammatory status of the oral cavity can have a profound impact on systemic health. In this review we discuss the disease states impacted by periodontitis and explore potential mechanisms whereby oral inflammation could promote loss of homeostasis at distant sites.

Amphiregulin-producing γδ T cells are vital for safeguarding oral barrier immune homeostasis.

γδ T cells are enriched at barrier sites such as the gut, skin, and lung, where their roles in maintaining barrier integrity are well established. However, how these cells contribute to homeostasis at the gingiva, a key oral barrier and site of the common chronic inflammatory disease periodontitis, has not been explored. Here we demonstrate that the gingiva is policed by γδ T cells with a T cell receptor (TCR) repertoire that diversifies during development. Gingival γδ T cells accumulated rapidly after birth in response to barrier damage, and strikingly, their absence resulted in enhanced pathology in murine models of the oral inflammatory disease periodontitis. Alterations in bacterial communities could not account for the increased disease severity seen in γδ T cell-deficient mice. Instead, gingival γδ T cells produced the wound healing associated cytokine amphiregulin, administration of which rescued the elevated oral pathology of tcrδ-/- mice. Collectively, our results identify γδ T cells as critical constituents of the immuno-surveillance network that safeguard gingival tissue homeostasis.

Tissue-resident macrophages in the intestine are long lived and defined by Tim-4 and CD4 expression.

A defining feature of resident gut macrophages is their high replenishment rate from blood monocytes attributed to tonic commensal stimulation of this site. In contrast, almost all other tissues contain locally maintained macrophage populations, which coexist with monocyte-replenished cells at homeostasis. In this study, we identified three transcriptionally distinct mouse gut macrophage subsets that segregate based on expression of Tim-4 and CD4. Challenging current understanding, Tim-4+CD4+ gut macrophages were found to be locally maintained, while Tim-4-CD4+ macrophages had a slow turnover from blood monocytes; indeed, Tim-4-CD4- macrophages were the only subset with the high monocyte-replenishment rate currently attributed to gut macrophages. Moreover, all macrophage subpopulations required live microbiota to sustain their numbers, not only those derived from blood monocytes. These findings oppose the prevailing paradigm that all macrophages in the adult mouse gut rapidly turn over from monocytes in a microbiome-dependent manner; instead, these findings supplant it with a model of ontogenetic diversity where locally maintained subsets coexist with rapidly replaced monocyte-derived populations.

Tissue-Specific Immunity at the Oral Mucosal Barrier.

The oral mucosal barrier is constantly exposed to a plethora of triggers requiring immune control, including a diverse commensal microbiome, ongoing damage from mastication, and dietary and airborne antigens. However, how these tissue-specific cues participate in the training of immune responsiveness at this site is minimally understood. Moreover, the mechanisms mediating homeostatic immunity at this interface are not yet fully defined. Here we present basic aspects of the oral mucosal barrier and discuss local cues that may modulate and train local immune responsiveness. We particularly focus on the immune cell network mediating immune surveillance at a specific oral barrier, the gingiva - a constantly stimulated and dynamic environment where homeostasis is often disrupted, resulting in the common inflammatory disease periodontitis.

D-mannose induces regulatory T cells and suppresses immunopathology.

D-mannose, a C-2 epimer of glucose, exists naturally in many plants and fruits, and is found in human blood at concentrations less than one-fiftieth of that of glucose. However, although the roles of glucose in T cell metabolism, diabetes and obesity are well characterized, the function of D-mannose in T cell immune responses remains unknown. Here we show that supraphysiological levels of D-mannose safely achievable by drinking-water supplementation suppressed immunopathology in mouse models of autoimmune diabetes and airway inflammation, and increased the proportion of Foxp3+ regulatory T cells (Treg cells) in mice. In vitro, D-mannose stimulated Treg cell differentiation in human and mouse cells by promoting TGF-ß activation, which in turn was mediated by upregulation of integrin αvß8 and reactive oxygen species generated by increased fatty acid oxidation. This previously unrecognized immunoregulatory function of D-mannose may have clinical applications for immunopathology.

Transforming Growth Factor-ß Signaling in Regulatory T Cells Controls T Helper-17 Cells and Tissue-Specific Immune Responses.

Regulatory T cells (Treg cells) perform suppressive functions in disparate tissue environments and against many inflammatory insults, yet the tissue-enriched factor(s) that influence Treg cell phenotype and function remain largely unknown. We have shown a vital role for transforming growth factor-ß (TGF-ß) signals in safe-guarding specific Treg cell functions. TGF-ß signals were dispensable for steady-state Treg cell homeostasis and for Treg cell suppression of T cell proliferation and T helper-1 (Th1) cell differentiation. However, Treg cells require TGF-ß signals to appropriately dampen Th17 cells and regulate responses in the gastrointestinal tract. TGF-ß signaling maintains CD103 expression, promotes expression of the colon-specific trafficking molecule GPR15, and inhibits expression of GPR174, a receptor for lysophosphatidylserine, on Treg cells, collectively supporting the accumulation and retention of Treg cells in the colon and control of colitogenic responses. Thus, we reveal an unrecognized function for TGF-ß signaling as an upstream factor controlling Treg cell activity in specific tissue environments.