IRF8-Dependent Type I Conventional Dendritic Cells (cDC1s) Control Post-Ischemic Inflammation and Mildly Protect Against Post-Ischemic Acute Kidney Injury and Disease

Post-ischemic acute kidney injury and disease (AKI/AKD) involve acute tubular necrosis and irreversible nephron loss. Mononuclear phagocytes including conventional dendritic cells (cDCs) are present during different phases of injury and repair, but the functional contribution of this subset remains controversial. Transcription factor interferon regulatory factor 8 (IRF8) is required for the development of type I conventional dendritic cells (cDC1s) lineage and helps to define distinct cDC1 subsets. We identified one distinct subset among mononuclear phagocyte subsets according to the expression patterns of CD11b and CD11c in healthy kidney and lymphoid organs, of which IRF8 was significantly expressed in the CD11blowCD11chigh subset that mainly comprised cDC1s. Next, we applied a Irf8-deficient mouse line (Irf8fl/flClec9acre mice) to specifically target Clec9a-expressing cDC1s in vivo. During post-ischemic AKI/AKD, these mice lacked cDC1s in the kidney without affecting cDC2s. The absence of cDC1s mildly aggravated the loss of living primary tubule and decline of kidney function, which was associated with decreased anti-inflammatory Tregs-related immune responses, but increased T helper type 1 (TH1)-related and pro-inflammatory cytokines, infiltrating neutrophils and acute tubular cell death, while we also observed a reduced number of cytotoxic CD8+ T cells in the kidney when cDC1s were absent. Together, our data show that IRF8 is indispensable for kidney cDC1s. Kidney cDC1s mildly protect against post-ischemic AKI/AKD, probably via suppressing tissue inflammation and damage, which implies an immunoregulatory role for cDC1s

Selective depletion of a CD64-expressing phagocyte subset mediates protection against toxic kidney injury and failure

Dendritic cells (DC), macrophages, and monocytes, collectively known as mononuclear phagocytes (MPs), critically control tissue homeostasis and immune defense. However, there is a paucity of models allowing to selectively manipulate subsets of these cells in specific tissues. The steady-state adult kidney contains four MP subsets with Clec9a-expression history that include the main conventional DC1 (cDC1) and cDC2 subtypes as well as two subsets marked by CD64 but varying levels of F4/80. How each of these MP subsets contributes to the different phases of acute kidney injury and repair is unknown. We created a mouse model with a Cre-inducible lox-STOP-lox-diphtheria toxin receptor cassette under control of the endogenous CD64 locus that allows for diphtheria toxin–mediated depletion of CD64-expressing MPs without affecting cDC1, cDC2, or other leukocytes in the kidney. Combined with specific depletion of cDC1 and cDC2, we revisited the role of MPs in cisplatin-induced kidney injury. We found that the intrinsic potency reported for CD11c$^{+}$cells to limit cisplatin toxicity is specifically attributed to CD64$^{+}$MPs, while cDC1 and cDC2 were dispensable. Thus, we report a mouse model allowing for selective depletion of a specific subset of renal MPs. Our findings in cisplatin-induced injury underscore the value of dissecting the functions of individual MP subsets in kidney disease, which may enable therapeutic targeting of specific immune components in the absence of general immunosuppression

Environmental signals rather than layered ontogeny imprint the function of type 2 conventional dendritic cells in young and adult mice

Conventional dendritic cells (cDC) are key activators of naive T cells, and can be targeted in adults to induce adaptive immunity, but in early life are considered under-developed or functionally immature. Here we show that, in early life, when the immune system develops, cDC2 exhibit a dual hematopoietic origin and, like other myeloid and lymphoid cells, develop in waves. Developmentally distinct cDC2 in early life, despite being distinguishable by fate mapping, are transcriptionally and functionally similar. cDC2 in early and adult life, however, are exposed to distinct cytokine environments that shape their transcriptional profile and alter their ability to sense pathogens, secrete cytokines and polarize T cells. We further show that cDC2 in early life, despite being distinct from cDC2 in adult life, are functionally competent and can induce T cell responses. Our results thus highlight the potential of harnessing cDC2 for boosting immunity in early life.</p

Recognition of apoptotic and necrotic cancer cells by macrophages

Makrofagi związane z nowotworami (TAMs) odgrywają kluczową rolę w kształtowaniu mikrośrodowiska wewnątrz guza litego. TAMs posiadają zarówno przeciw- jak i pro-nowotworowe funkcje, polegające na rozpoznaniu i fagocytowaniu komórek nowotworowych oraz produkcji cytokin w odpowiedzi na sygnały docierające się z mikrośrodowiska. Rozpoznanie i fagocytoza komórek apoptotycznych są stosunkowo dobrze opisane, jednak wiedza na temat rozpoznania innych typów śmierci komórki jest nadal ograniczona. Niniejsze badania skupiały się na indukcji apoptozy/nekrozy w komórkach nowotworowych (A498) pochodzących z raka nerki oraz określeniu wpływu martwych komórek nowotworowych na makrofagi. W celu indukcji apoptozy/nekrozy komórki nowotworowe hodowano w warunkach naśladujących mikrośrodowisko wewnątrz guza litego, a także traktowano inhibitorem HSP90, 17-DMAG. Kolejnym krokiem badań było określenie wpływu apoptotycznych/nekrotycznych komórek nowotworowych na makrofagi. W tym celu makrofagi stymulowano martwymi komórkami nowotworowymi oraz mierzono produkcję podstawowych cytokin w odpowiedzi na nie.Na podstawie przeprowadzonych badań uznano, iż martwe komórki nowotworowe nie wywierają prozapalnego efektu na makrofagi oraz znoszą stan zapalny wywołany LPSem. Przedstawione wyniki sugerują, iż martwe komórki nowotworowe biorą czynny udział w procesie różnicowania się makrofagów w kierunku przeciw-zapalnym oraz kształtowaniu immunosupresyjnego mikrośrodowiska wewnątrz guza litego.Tumor-associated macrophages (TAMs) play a crucial role in microenvironment inside solid tumors. TAMs have both anti-and pro-tumor functions. They recognize and uptake tumors cells and produce cytokines in response to signals from microenvironment. Although the recognition and phagocytosis of apoptotic cells have been extensively described, little is known about recognition of other types of cell death cells.The aim of the study was to induce apoptosis/necrosis in cancer cells from kidney carcinoma (A498) and to identify the influence of dead tumor cells on macrophages. Cancer cells were cultured under conditions that mimic microenvironment inside solid tumors, and treated with the inhibitor of HSP90, 17-DMAG, for induction of apoptosis/necrosis. The next step of the study was to determine the influence of apoptotic/necrotic cancer cells on macrophages. For this purpose, macrophages were stimulated with dead cancer cells and the production of cytokines was measured.Taken together the data indicate that the dead cancer cells did not induce pro-inflammatory response in macrophages; moreover, the dead cancer cells abolished the pro-inflammatory response to LPS. Consequently, it has been suggested, that dead cancer cells are involved in anti-inflammatory differentiation of macrophages and promotion of immunosuppressive microenvironment inside solid tumors

Infection with <i>Leishmania major</i> results in enhanced phagocytosis of apoptotic cells by neutrophils.

<p>(A) A micrograph of Diff Quik-stained neutrophils infected with <i>L</i>. <i>major</i> promastigotes. Arrows indicate <i>L</i>. <i>major</i> parasites inside neutrophils. (B) Percentage of infected neutrophils after co-incubation with <i>L</i>. <i>major</i> for four hours. (C and D) Representative flow cytometry dot plots and histograms of (C) non-infected and (D) <i>L</i>. <i>major</i>-infected PKH-67-labeled neutrophils co-incubated with PKH-26-labeled apoptotic cells. (E) Phagocytosis of apoptotic cells by non-infected and <i>L</i>. <i>major</i>-infected neutrophils in the absence or presence of cytochalasin D. (F) Phagocytosis of latex beads by non-infected and <i>L</i>. <i>major</i>-infected neutrophils. Data are shown as mean ± SEM of five (B, E) or three (E) independent experiments; *** p ≤ 0.001, ns = not significant.</p

Enhanced survival of <i>Leishmania major</i> in neutrophil granulocytes in the presence of apoptotic cells

<div><p>Neutrophil granulocytes are the first leukocytes that encounter and phagocytose <i>Leishmania major</i> (<i>L</i>. <i>major</i>) parasites in the infected skin. The parasites can nonetheless survive within neutrophils. However, the mechanisms enabling the survival of <i>Leishmania</i> within neutrophils are still elusive. Previous findings indicated that human neutrophils can engulf apoptotic cells. Since apoptotic neutrophils are abundant in infected tissues, we hypothesized that the uptake of apoptotic cells results in diminished anti-leishmanial activity and, consequently, contributes to enhanced survival of the parasites at the site of infection. In the present study, we demonstrated that <i>L</i>. <i>major</i>-infected primary human neutrophils acquire enhanced capacity to engulf apoptotic cells. This was associated with increased expression of the complement receptors 1 and 3 involved in phagocytosis of apoptotic cells. Next, we showed that ingestion of apoptotic cells affects neutrophil antimicrobial functions. We observed that phagocytosis of apoptotic cells by neutrophils downregulates the phosphorylation of p38 MAPK and PKCδ, the kinases involved in activation of NADPH oxidase and hence reactive oxygen species (ROS) production. In line, uptake of apoptotic cells inhibits TNF- and <i>L</i>. <i>major</i>-induced ROS production by neutrophils. Importantly, we found that the survival of <i>Leishmania</i> in neutrophils is strongly enhanced in neutrophils exposed to apoptotic cells. Together, our findings reveal that apoptotic cells promote <i>L</i>. <i>major</i> survival within neutrophils by downregulating critical antimicrobial functions. This suggests that the induction of enhanced uptake of apoptotic cells represents a novel evasion mechanism of the parasites that facilitates their survival in neutrophil granulocytes.</p></div

Co-incubation with apoptotic cells results in diminished TNF-induced phosphorylation of PKCδ and p38-MAPK in neutrophils.

<p>Freshly isolated neutrophils were incubated in the absence or presence of apoptotic cells for 90 min prior to stimulation with TNF-α for 15 min, and the phosphorylation of p38-MAPK and PKCδ was assessed by western blot. A representative blot of phosphorylated p38-MAPK (A) and PKCδ (C) are shown. Data were quantified and normalized to β-actin (B and D). Data are shown as mean ± SEM of three-four independent experiments; *p ≤ 0.05, **p ≤ 0.01.</p

Apoptotic cells modulate the course of <i>L</i>. <i>major</i> infection in neutrophils.

<p>(1) <i>Leishmania</i> promastigotes are recognized by neutrophils; (2) <i>Leishmania</i>-infected neutrophils activate antimicrobial signaling pathways responsible for generation of ROS; (3) Signals from apoptotic cells suppress phosphorylation of pro-inflammatory kinases and inhibit ROS production in neutrophils leading to improved survival of the parasites; (4) <i>Leishmania</i> can upregulate neutrophil-mediated phagocytosis of apoptotic cells to reduce microbicidal environment within neutrophils.</p

Complement factors are necessary for efficient neutrophil-mediated phagocytosis of apoptotic cells.

<p>Neutrophils were co-incubated with apoptotic cells in the presence of 30% normal human serum, C1q-depleted or C3-deficient serum with or without reconstitution of the deficient sera with exogenous C1q or C3, respectively. Phagocytosis of apoptotic cells was assessed by flow cytometry. Data are shown as mean ± SEM of four independent experiments (A). Neutrophils were infected with <i>L</i>. <i>major</i> promastigotes (ratio of 1 to 10) for 4 h at 37°C. Surface expression of CD11b (B), CD35 (C), CD11c (D), CD14 (E), and CD44 (F) was assessed by flow cytometry. Data are shown as mean ± SEM of three to seven independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, ns = not significant.</p