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On macrophage contributions to tissue homeostasis: New insights on pancreas development and healing of ischemic injury
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

Besides providing host defence against innumerable threats, macrophages display additional key functions for preservation of tissue homeostasis. This thesis includes four studies that explore novel macrophage functions in both the development of islets of Langerhans and healing of ischemic injury in mice.

The aim of Study I was to explore the involvement of pancreatic macrophages in postnatal islet development. We found that neonatal pancreas contained high density of macrophages. Neonatal infections reduced the number of pancreatic macrophages transiently and resulted in both impaired β cell maturation and associated long-standing glucose intolerance. Moreover, clodronate depletion of pancreatic macrophages in the neonate also resulted in long-standing impairment of glucose handling. Together, these results demonstrate that macrophages in the neonatal pancreas are important for maturation of islet function.

We then wanted to understand how macrophages contribute to healing of ischemic injury based on the observation that they accumulate at perivascular positions following ischemia. We found that blood flow at the site of ischemia was regulated by perivascular macrophages in an iNOS-dependent manner, which could be targeted to increase tissue healing (Study II). Next, we investigated if these perivascular macrophages trans-differentiate into mural cells. By lineage tracing, we found that macrophages undergo a phenotype shift at the site of ischemic injury, as they down-regulated the expression of myeloid cell lineage markers (CD45/CX3CR1/CD11b) and upregulated the expression of the mural cell marker PDGFRβ (Study III). Lastly, we addressed if macrophages are involved in vascular remodelling important for tissue normalization by pruning excessive vessels at the site of injury. Indeed, MMR+-macrophages were found to support vessel pruning during vascular normalization at late phases of healing (Study IV).

In conclusion, this thesis reveals novel functions of macrophages as they support postnatal maturation of the insulin-producing β cells of the pancreas, as well as restore blood flow and normalize the vasculature during healing of ischemic injuries. Together, the studies in this thesis contribute to illustrating the ample and diverse macrophage curriculum and how macrophage skills cooperate to ensure homeostasis.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 82
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1605
Keywords [en]
macrophages, islets of Langerhans, β cell function, postnatal β cell maturation, hindlimb ischemia, blood flow regulation, mural cells, vessel pruning, diabetes mellitus, ischemic diseases
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-395535ISBN: 978-91-513-0787-9 (print)OAI: oai:DiVA.org:uu-395535DiVA, id: diva2:1362488
Public defence
2019-12-06, Room B41, BMC, Hursagatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2019-11-15 Created: 2019-10-21 Last updated: 2019-11-15
List of papers
1. Neonatal infections impair islet maturation and long-term glucose homeostasis by disturbing the pancreatic macrophage population.
Open this publication in new window or tab >>Neonatal infections impair islet maturation and long-term glucose homeostasis by disturbing the pancreatic macrophage population.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The development of the pancreatic islet occurs late during pregnancy and continues in the neonate with β cell development of functional maturity. Here we show that pancreatic macrophages are present in highest densities in the neonate. In addition, we found that transient neonatal infections reduced the numbers of pancreatic macrophages and resulted in development of impaired glucose tolerance that persisted into adolescence when the infection was cleared and macrophage levels were restored (three- and six-week-old mice). Reduced islet insulin content and β cell proliferation, together with decreased proportion of MafA+ β cells, were observed in six-week-old mice subjected to neonatal infections. Transient neonatal depletion of macrophages recapitulated the effects of neonatal infections: longstanding impairment of glucose homeostasis and reduced β cell proliferation (six-week-olds), indicating a role for macrophages in postnatal islet maturation. Our findings uncover a novel function for macrophages in securing appropriate postnatal islet development. Immune challenges occurring at this time distracted the pancreatic macrophage population and hindered islet maturation, thereby resulting in longstanding impairment of glucose homeostasis. 

Keywords
Macrophages, postnatal pancreas development, β cell maturation, infections, glucose homeostasis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-395529 (URN)
Available from: 2019-10-20 Created: 2019-10-20 Last updated: 2019-10-27
2. Perivascular macrophages regulate blood flow following tissue damage
Open this publication in new window or tab >>Perivascular macrophages regulate blood flow following tissue damage
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Ischemic injuries remain a leading cause of mortality and morbidity world-wide, and restoration of functional blood perfusion is vital to limit tissue damage and support healing. Sterile inflammation is a hallmark of ischemic injury, as macrophages and neutrophils accumulate to clear tissue debris and support vascular regrowth. Here, we delineate the role of macrophages in reestablishment of functional tissue perfusion following ischemic injury and design an immunotherapeutic approach to improve tissue restoration.

Methods: Macrophage-endothelial cell interactions were assessed by intravital microscopy of ischemic hindlimb muscle in CX3CR1GFP/+ mice, and by confocal microscopy of human tissues from amputated legs. Functional muscle perfusion was evaluated as the response to increased local tissue temperature (Δ10°C, heat-induced hyperemia) and measured by Laser Doppler Flowmetry and Laser Speckle Analysis in wild-type and transgenic mice with inducible conditional deletion of inducible nitric oxide synthase (iNOS) in macrophages (Cx3cr1-CreER;Nos2fl/fl, LysM-Cre;Nos2fl/fl). To target restorative functions of macrophages in ischemic muscles, local chemokine overexpression was induced by designed chemokine-encoding DNA plasmids.

Results: We found that macrophages accumulate at perivascular positions along blood vessels in ischemic muscles from both humans and mice, where they express high levels of iNOS. Further, we demonstrated that the hyperemic response to heat was shifted from eNOS (endothelial)-dependence in healthy mouse muscles to completely rely on iNOS in ischemic muscle. Specific depletion of iNOS in macrophages did not affect vascular architecture but highly compromised the ability to upregulate blood flow in the ischemic muscle, which resulted in aggravated ischemic damage. The macrophages in ischemic muscles expressed high levels of CXCR4 and CCR2, and local overexpression of the corresponding chemokine CXCL12 but not CCL2 increased macrophage numbers and perivascular positioning in the ischemic muscle. As a result, CXCL12-overexpression increased the number of perfused blood vessels, improved functional muscle perfusion in an iNOS- and macrophage-dependent manner, and ultimately restored limb function.

Conclusions: This study establishes a new function for macrophages during tissue repair, as they regulate blood flow through the release of iNOS-produced NO. Further, we demonstrate that macrophages can be therapeutically targeted to improve blood flow regulation and functional recovery of ischemic tissues.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-395532 (URN)
Note

         * Indicates equal contribution

Available from: 2019-10-20 Created: 2019-10-20 Last updated: 2019-10-21
3. Macrophages adopt mural cell function and marker expression following ischemia
Open this publication in new window or tab >>Macrophages adopt mural cell function and marker expression following ischemia
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

In response to tissue ischemia, angiogenesis is initiated and macrophages accumulate in perivascular positions. Mural cells (pericytes and smooth muscle cells) will in this process also attain a perivascular position and mediate blood vessel maturation and blood flow regulation. With fate mapping of macrophages following hindlimb ischemia in mouse models, we show that ischemia-recruited perivascular macrophages down-regulate the expression of the myeloid cell lineage markers CD45, CX3CR1, and CD11b and upregulate expression of the mural cell markers PDGFRβ. Depletion of macrophages in the context of ischemia resulted in severe mural cell deficiency in newly formed vessels and impaired blood vessel maturation.  Taken together, we here demonstrate that macrophages are crucial for blood vessel maturation following ischemic injury by adopting a mural cell identity. 

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-395533 (URN)
Note

* Indicates equal contribution

Available from: 2019-10-20 Created: 2019-10-20 Last updated: 2019-10-21
4. Macrophages contribute to vessel normalization during healing of ischemic injury
Open this publication in new window or tab >>Macrophages contribute to vessel normalization during healing of ischemic injury
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Physiological regression of superfluous micro-vessels, vascular pruning, is observed during formation of functional vascular networks in fetal development. The current study investigates the vascular dynamics during healing of injured adult tissues in the mouse model of hind limb ischemia. We found that prompt angiogenesis that occurs in M. gastrocnemius to compensate for loss of tissue perfusion, results in blood vessel densities that are higher than those found in healthy muscles. Vessel density peaked at day 14 post ischemia to thereafter recede to normal levels by day 21, indicating that vessel pruning occurs during healing to ensure establishment of an optimal vascular tree. Macrophages are professional phagocytes and we found them present in high numbers at sites of ischemic injury, where they were positioned in close contact with the vascular network and displayed phagocytic activity at the time of vessel pruning. Interestingly, macrophage depletion between day 14 and 21 post-ischemia resulted in reduced vessel regression. Indeed, by using a reporter mouse for endothelial cells, we found that macrophages engulf endothelial cells at the ischemic site at this time point. Taken together, our results indicate a role for macrophages in vessel normalization by pruning superfluous vasculatur segments that form initially during healing of ischemic injuries. 

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-395534 (URN)
Available from: 2019-10-20 Created: 2019-10-20 Last updated: 2019-10-21

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