Development of macrophages in the intestine

Macrophages (mφ) are one the most numerous leukocytes present in the healthy gut and contribute to both harmful and beneficial immune reactions. In the colon, mφ are exposed continuously to large amounts of material from the environment, including harmful agents such as invasive bacteria, viruses and parasites, as well as harmless materials such as food proteins and the commensal bacteria which inhabit the healthy intestine. As a result, mφ play an important role in helping defend the intestine against harmful invaders. However if these cells make similar reactions to harmless food proteins or commensal bacteria, it would be both wasteful and detrimental, likely leading to inflammatory diseases such as coeliac disease and Crohn’s disease. Several genes, which underlie susceptibility to Crohn’s disease are involved in controlling how macrophages respond to the microbiota, with considerable evidence indicating that this reflects a loss of the normal unresponsiveness that characterises intestinal macrophages in the healthy intestine. One of the most significant aspects of the epidemiology of Crohn’s disease is a particularly rapid increase in its incidence in childhood, suggesting that the first encounters between the microbiota and intestinal macrophages may be of critical importance in determining disease susceptibility. Given this link, it is essential that we elucidate the processes controlling macrophage seeding and development in the intestine and this was an aim of this thesis. In the adult healthy colon, two main mφ subsets can be identified: A dominant and homogenous one, made up of mature mφ, which express high levels of F4/80, MHC II, CX3CR1, are CD11bint/+, highly phagocytic and produce high amounts of IL10. The second mφ group is relatively smaller and is much more heterogeneous. These cells express intermediate levels of F4/80 and CX3CR1, are CD11b+ and can be divided into 3 subsets based on their levels of Ly6C and MHC II. These subsets represent a maturation continuum towards the mature mφ phenotype. Recent reports have suggested that resident macrophages in healthy tissues may be derived from yolk-sac and/or foetal liver precursors that seed tissues during development and subsequently self-renew locally. In contrast, it is proposed that macrophages in inflammation are generated by recruitment of blood monocytes, raising the possibility that these different origins could be exploited in therapy. However none of these studies have examined macrophages in the intestine and recent work in our laboratory has suggested that monocytes may be the precursors of macrophages in both healthy and inflamed gut of adult mice. Therefore, the aims of this thesis were to investigate the development of murine colonic mφ from birth until adulthood, examining the relative roles of the yolk sac, foetal liver and bone marrow monocytes, exploring their functions and comparing them with the well-characterised adult mφ. In addition, I also examined how mφ phenotype and functions are influenced by the microbiota using broad-spectrum antibiotics and germ free mice. Lastly, I examined the role of fractalkine and its receptor CX3CR1 in defining the development and functions of intestinal macrophages. Development of macrophages in early life The initial characterisation and comparison of colonic mφ subsets is included in Chapter 3. In this chapter, I describe a series of experiments adapting existing protocols and techniques used for examining the adult murine intestine in order to analyse the origin, phenotype and functions of murine colonic macrophages from late foetal life through to adulthood. These studies found that intestinal mφ are present before birth, with similar levels of phagocytic ability and IL10, TNFα and CD163 mRNA expression to the adult. However, the numbers and phenotype of mφ in the intestine do not reach the adult level until the 3rd week of postnatal life. This phenomenon appears to reflect the de novo recruitment of blood monocytes in a CCR2-dependent fashion at this time and throughout adult life, but not at early stages of life. In the colon of newborn mice, two macrophage populations can be observed and are clearly differentiated based on their F4/80 and CD11b expression: F4/80hi CD11bint/+ and F4/80lo CD11b+. Interestingly, unlike adult colonic F4/80hi mφ, the majority of F4/80hi neonatal cells do not express MHC II, however they gradually express this molecule as they age. In addition to acquiring MHC II expression, the two populations in the newborn colon gradually merge and from the 3rd week of life it is difficult to discriminate them reliably. My experiments show that both mφ subsets proliferate actively during the first 2 weeks of life, but this is later reduced and maintained at low levels indicating that there is no self-renewal of mature mφ. Moreover, fate-mapping analysis carried out in collaboration with Professor Frederic Geissmann, showed that yolk sac-derived precursors contribute only minimally to the pool of colonic mφ, even at early life stages. Conversely, additional fate mapping studies suggested that most intestinal macrophages are derived from Flt3+ progenitors. Taken together, the results in this chapter demonstrate that blood monocytes are vital in replenishing the intestinal macrophage pool in the steady state, setting them apart from other tissue macrophages, which derive from primitive progenitors. Investigating the effect of the microbiota on intestinal macrophage subsets In Chapter 4, I assessed the effects of the commensal microbiota on intestinal mφ, using two different approaches: First, I assessed the function and gene expression of colonic macrophages following administration of broad-spectrum antibiotics. My results showed that this did not alter the numbers, phenotype, intracellular cytokine production or mRNA expression by macrophages. Several reasons may account for this, including dose/nature of antibiotics, length of administration or lifespan of macrophages. To overcome these issues, I compared the phenotype of colonic mφ in germ free (GF) and conventionally (CNV) reared mice of different ages in collaboration with Dr David Artis. Absolute absence of microbiota in GF mice severely impacted Ly6Chi monocyte recruitment to the colon, suggesting that constant recruitment of monocytes to the gut is at least in part due to the microbial burden. The biggest differences between GF and CNV mice were evident at 3 weeks of age, when GF mice had a much lower number and frequency of monocyte-derived cells than their CNV counterparts. By 12 weeks of age, Ly6Chi mφ populations from GF mice were partially restored, although the expression of MHC II by F4/80hi mφ remained reduced. Additionally, I FACS-purified F4/80hi cells from GF and CNV adults and sent RNA for microarray analysis, the results of which we are waiting to receive. This data will provide further information regarding how GF intestinal mφ differ from those found in conventional animals. Role of the CX3CL1-CX3CR1 axis in mφ development and function As mature colonic mφ express high levels of the chemokine receptor CX3CR1 (fractalkine), finally, in Chapter 5 I went on to investigate the role of CX3CL1-CX3CR1 axis in colonic lamina propria. In addition to the high expression of CX3CR1 by colonic mφ, its ligand, CX3CL1 has been reported to be expressed at high levels by the intestinal epithelium. Furthermore, as there is strong evidence that the CX3CL1-CX3CR1 axis may be involved in inflammation in several tissues, we hypothesised this axis might play a role in mφ function in the gut. To this end, I examined mφ phenotype, activation status and survival following in vitro co-culture of WT or CX3CR1-deficient bone marrow-derived mφ with an epithelial cell line modified to express either the soluble or membrane-bound forms of CX3CL1. I also examined the development of chemically induced colitis in CX3CR1-deficient mice. Finally, since it has been reported by the lab of Oliver Pabst, that the lack of CX3CR1 results in reduced IL10 production by intestinal mφ, I compared the ability of WT and CX3CR1-deficient mice to prime T cells after being fed with ovalbumin together with an adjuvant. The results from this chapter failed to show any definitive role of the CX3CL1-CX3CR1 axis in mφ function in either the steady state or in the setting of inflammation. My in vitro studies did not show any significant difference between WT and CX3CR1 deficient intestinal mφ in terms of survival, or co-stimulatory molecule expression, nor did bone marrow mφ (BMM) from CX3CR1 KO mice show differences in co-stimulatory molecules and pro-inflammatory cytokine production with or without stimulation by LPS. Moreover, the responses of wild type BMM were not altered by exposure to exogenous CX3CL1 either in soluble form, or when expressed as a transmembrane form by epithelial cells. The in vivo assessment of CX3CR1 during inflammation, Ly6Chi CX3CR1int cells increased after 4 days on DSS, however, the lack of CX3CR1 failed to confer protection from colitis in a consistent manner, suggesting that there may be more factors responsible for colonic inflammation apart from the CX3CL1-CX3CR1 axis. Taken together, the results of this thesis highlight that important cellular changes take place during the development of mφ in the intestine. In addition, the presence or absence of microbiota plays a crucial role in this development with acquisition of MHC II depending at least in part on the presence of microbes. Microarray data obtained from purified F4/80hi mφ populations of GF and CNV mice may reveal interesting differences and suggest how mφ phenotype and function may be regulated by the microbiota. Finally, I have shown that the CX3CL1-CX3CR1 axis plays a redundant role in the regulation of intestinal mφ phenotype and function with mφ from CX3CR1-deficient animals appearing to function normally in both health and disease

Regulatory T cells control the dynamic and site-specific polarization of total CD4 T cells following Salmonella infection

FoxP3+ regulatory T cells (Tregs) control inflammation and maintain mucosal homeostasis, but their functions during infection are poorly understood. Th1, Th2, and Th17 cells can be identified by master transcription factors (TFs) T-bet, GATA3, and RORγT; Tregs also express these TFs. While T-bet+ Tregs can selectively suppress Th1 cells, it is unclear whether distinct Treg populations can alter Th bias. To address this, we used Salmonella enterica serotype Typhimurium to induce nonlethal colitis. Following infection, we observed an early colonic Th17 response within total CD4 T cells, followed by a Th1 bias. The early Th17 response, which contains both Salmonella-specific and non-Salmonella-specific cells, parallels an increase in T-bet+ Tregs. Later, Th1 cells and RORγT+ Tregs dominate. This reciprocal dynamic may indicate that Tregs selectively suppress Th cells, shaping the immune response. Treg depletion 1–2 days post-infection shifted the early Th17 response to a Th1 bias; however, Treg depletion 6–7 days post-infection abrogated the Th1 bias. Thus, Tregs are necessary for the early Th17 response, and for a maximal Th1 response later. These data show that Tregs shape the overall tissue CD4 T cell response and highlight the potential for subpopulations of Tregs to be used in targeted therapeutic approaches

Mucosal CD8 T Cell Responses Are Shaped by Batf3-DC After Foodborne Listeria monocytogenes Infection

While immune responses have been rigorously examined after intravenous Listeria monocytogenes (Lm) infection, less is understood about its dissemination from the intestines or the induction of adaptive immunity after more physiologic models of foodborne infection. Consequently, this study focused on early events in the intestinal mucosa and draining mesenteric lymph nodes (MLN) using foodborne infection of mice with Lm modified to invade murine intestinal epithelium (InlAM Lm). InlAM Lm trafficked intracellularly from the intestines to the MLN and were associated with Batf3-independent dendritic cells (DC) in the lymphatics. Consistent with this, InlAM Lm initially disseminated from the gut to the MLN normally in Batf3–/– mice. Activated migratory DC accumulated in the MLN by 3 days post-infection and surrounded foci of InlAM Lm. At this time Batf3–/– mice displayed reduced InlAM Lm burdens, implicating cDC1 in maximal bacterial accumulation in the MLN. Batf3–/– mice also exhibited profound defects in the induction and gut-homing of InlAM Lm-specific effector CD8 T cells. Restoration of pathogen burden did not rescue antigen-specific CD8 T cell responses in Batf3–/– mice, indicating a critical role for Batf3 in generating anti-InlAM Lm immunity following foodborne infection. Collectively, these data suggest that DC play diverse, dynamic roles in the early events following foodborne InlAM Lm infection and in driving the establishment of intestinal Lm-specific effector T cells.Fil: Imperato, Jessica Nancy. Stony Brook University Renaissance School Of Medicine; Estados UnidosFil: Xu, Daqi. Uconn Health; Estados UnidosFil: Romagnoli, Pablo Alberto. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigacion Medica Mercedes y Martin Ferreyra. Grupo Vinculado Centro de Investigacion En Medicina Traslacional Severo R. Amuchastegui - Cimetsa | Universidad Nacional de Cordoba. Instituto de Investigacion Medica Mercedes y Martin Ferreyra. Grupo Vinculado Centro de Investigacion En Medicina Traslacional Severo R. Amuchastegui - Cimetsa | Instituto de Investigacion Medica Mercedes y Martin Ferreyra. Instituto de Investigacion Medica Mercedes y Martin Ferreyra. Grupo Vinculado Centro de Investigacion En Medicina Traslacional Severo R. Amuchastegui - Cimetsa.; ArgentinaFil: Qiu, Zhijuan. Stony Brook University Renaissance School Of Medicine; Estados UnidosFil: Perez, Pedro. Stony Brook University Renaissance School Of Medicine; Estados UnidosFil: Khairallah, Camille. Stony Brook University Renaissance School Of Medicine; Estados UnidosFil: Pham, Quynh Mai. Uconn Health; Estados UnidosFil: Andrusaite, Anna. University of Glasgow; Reino UnidoFil: Bravo Blas, Alberto. The Beatson Institute For Cancer Research; Reino UnidoFil: Milling, Simon W. F.. University of Glasgow; Reino UnidoFil: Lefrancois, Leo. Uconn Health; Estados UnidosFil: Khanna, Kamal M.. University of New York; Estados UnidosFil: Puddington, Lynn. Uconn Health; Estados UnidosFil: Sheridan, Brian S.. Stony Brook University Renaissance School Of Medicine; Estados Unido

Antibiotics induce sustained dysregulation of intestinal T cell immunity by perturbing macrophage homeostasis

Macrophages in the healthy intestine are highly specialized and usually respond to the gut microbiota without provoking an inflammatory response. A breakdown in this tolerance leads to inflammatory bowel disease (IBD), but the mechanisms by which intestinal macrophages normally become conditioned to promote microbial tolerance are unclear. Strong epidemiological evidence linking disruption of the gut microbiota by antibiotic use early in life to IBD indicates an important role for the gut microbiota in modulating intestinal immunity. Here, we show that antibiotic use causes intestinal macrophages to become hyperresponsive to bacterial stimulation, producing excess inflammatory cytokines. Re-exposure of antibiotic-treated mice to conventional microbiota induced a long-term, macrophage-dependent increase in inflammatory T helper 1 (T 1) responses in the colon and sustained dysbiosis. The consequences of this dysregulated macrophage activity for T cell function were demonstrated by increased susceptibility to infections requiring T 17 and T 2 responses for clearance (bacterial and helminth infections), corresponding with increased inflammation. Short-chain fatty acids (SCFAs) were depleted during antibiotic administration; supplementation of antibiotics with the SCFA butyrate restored the characteristic hyporesponsiveness of intestinal macrophages and prevented T cell dysfunction. Butyrate altered the metabolic behavior of macrophages to increase oxidative phosphorylation and also promoted alternative macrophage activation. In summary, the gut microbiota is essential to maintain macrophage-dependent intestinal immune homeostasis, mediated by SCFA-dependent pathways. Oral antibiotics disrupt this process to promote sustained T cell-mediated dysfunction and increased susceptibility to infections, highlighting important implications of repeated broad-spectrum antibiotic use

The mannose receptor (CD206) identifies a population of colonic macrophages in health and inflammatory bowel disease

To understand the contribution of mononuclear phagocytes (MNP), which include monocyte-derived intestinal macrophages, to the pathogenesis of inflammatory bowel disease (IBD), it is necessary to identify functionally-different MNP populations. We aimed to characterise intestinal macrophage populations in patients with IBD. We developed 12-parameter flow cytometry protocols to identify and human intestinal MNPs. We used these protocols to purify and characterize colonic macrophages from colonic tissue from patients with Crohn’s disease (CD), ulcerative colitis (UC), or non-inflamed controls, in a cross-sectional study. We identify macrophage populations (CD45+CD64+ HLA-DR+) and describe two distinct subsets, differentiated by their expression of the mannose receptor, CD206. CD206+ macrophages expressed markers consistent with a mature phenotype: high levels of CD68 and CD163, higher transcription of IL-10 and lower expression of TREM1. CD206− macrophages appear to be less mature, with features more similar to their monocytic precursors. We identified and purified macrophage populations from human colon. These appear to be derived from a monocytic precursor with high CCR2 and low CD206 expression. As these cells mature, they acquire expression of IL-10, CD206, CD63, and CD168. Targeting the newly recruited monocyte-derived cells may represent a fruitful avenue to ameliorate chronic inflammation in IBD

Monocytes mediate Salmonella Typhimurium-induced tumor growth inhibition in a mouse melanoma model

The use of bacteria as an alternative cancer therapy has been reinvestigated in recent years. SL7207: an auxotrophic Salmonella enterica serovar Typhimurium aroA mutant with immune-stimulatory potential has proven a promising strain for this purpose. Here, we show that systemic administration of SL7207 induces melanoma tumor growth arrest in vivo, with greater survival of the SL7207-treated group compared to control PBS-treated mice. Administration of SL7207 is accompanied by a change in the immune phenotype of the tumor-infiltrating cells toward pro-inflammatory, with expression of the TH1 cytokines IFN-γ, TNF-α, and IL-12 significantly increased. Interestingly, Ly6C+MHCII+ monocytes were recruited to the tumors following SL7207 treatment and were pro-inflammatory. Accordingly, the abrogation of these infiltrating monocytes using clodronate liposomes prevented SL7207-induced tumor growth inhibition. These data demonstrate a previously unappreciated role for infiltrating inflammatory monocytes underlying bacterial-mediated tumor growth inhibition. This information highlights a possible novel role for monocytes in controlling tumor growth, contributing to our understanding of the immune responses required for successful immunotherapy of cancer

Pancreatic metastases from renal cell carcinoma. Postoperative outcome after surgical treatment in a Spanish multicenter study (PANMEKID)

Background: Renal Cell Carcinoma (RCC) occasionally spreads to the pancreas. The purpose of our study is to evaluate the short and long-term results of a multicenter series in order to determine the effect of surgical treatment on the prognosis of these patients. Methods: Multicenter retrospective study of patients undergoing surgery for RCC pancreatic metastases, from January 2010 to May 2020. Variables related to the primary tumor, demographics, clinical characteristics of metastasis, location in the pancreas, type of pancreatic resection performed and data on short and long-term evolution after pancreatic resection were collected. Results: The study included 116 patients. The mean time between nephrectomy and pancreatic metastases' resection was 87.35 months (ICR: 1.51-332.55). Distal pancreatectomy was the most performed technique employed (50 %). Postoperative morbidity was observed in 60.9 % of cases (Clavien-Dindo greater than IIIa in 14 %). The median follow-up time was 43 months (13-78). Overall survival (OS) rates at 1, 3, and 5 years were 96 %, 88 %, and 83 %, respectively. The disease-free survival (DFS) rate at 1, 3, and 5 years was 73 %, 49 %, and 35 %, respectively. Significant prognostic factors of relapse were a disease free interval of less than 10 years (2.05 [1.13-3.72], p 0.02) and a history of previous extrapancreatic metastasis (2.44 [1.22-4.86], p 0.01). Conclusions: Pancreatic resection if metastatic RCC is found in the pancreas is warranted to achieve higher overall survival and disease-free survival, even if extrapancreatic metastases were previously removed. The existence of intrapancreatic multifocal compromise does not always warrant the performance of a total pancreatectomy in order to improve survival. (C) 2021 The Authors. Published by Elsevier Ltd

Repeated pancreatic resection for pancreatic metastases from renal cell Carcinoma: A Spanish multicenter study (PANMEKID)

Background and objectives: Recurrent isolated pancreatic metastasis from Renal Cell Carcinoma (RCC) after pancreatic resection is rare. The purpose of our study is to describe a series of cases of relapse of pancreatic metastasis from renal cancer in the pancreatic remnant and its surgical treatment with a repeated pancreatic resection, and to analyse the results of both overall and disease -free survival. Methods: Multicenter retrospective study of patients undergoing pancreatic resection for RCC pancreatic metastases, from January 2010 to May 2020. Patients were grouped into two groups depending on whether they received a single pancreatic resection (SPS) or iterative pancreatic resection. Data on short and long-term outcome after pancreatic resection were collected. Results: The study included 131 pancreatic resections performed in 116 patients. Thus, iterative pancreatic surgery (IPS) was performed in 15 patients. The mean length of time between the first pancreatic surgery and the second was 48.9 months (95 % CI: 22.2-56.9). There were no differences in the rate of postoperative complications. The DFS rates at 1, 3 and 5 years were 86 %, 78 % and 78 % vs 75 %, 50 % and 37 % in the IPS and SPS group respectively (p = 0.179). OS rates at 1, 3, 5 and 7 years were 100 %, 100 %, 100 % and 75 % in the IPS group vs 95 %, 85 %, 80 % and 68 % in the SPS group (p = 0.895). Conclusion: Repeated pancreatic resection in case of relapse of pancreatic metastasis of RCC in the pancreatic remnant is justified, since it achieves OS results similar to those obtained after the first resection

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research