Local macrophage proliferation, rather than recruitment from the blood, is a signature of TH2 inflammation

Abstract

A defining feature of inflammation is the accumulation of innate immune cells in the tissue that are thought to be recruited from the blood. We reveal that a distinct process exists in which tissue macrophages undergo rapid in situ proliferation in order to increase population density. This inflammatory mechanism occurred during T helper 2 (T(H)2)-related pathologies under the control of the archetypal T(H)2 cytokine interleukin-4 (IL-4) and was a fundamental component of T(H)2 inflammation because exogenous IL-4 was sufficient to drive accumulation of tissue macrophages through self-renewal. Thus, expansion of innate cells necessary for pathogen control or wound repair can occur without recruitment of potentially tissue-destructive inflammatory cells.

Fig. 1

Alternatively activated macrophages accumulate independently of blood monocytes. (A) Proportion of pleural cavity macrophages staining positive for RELMα following infection with L. sigmodontis and representative flow cytograms gated on CD11b⁺ Siglec-F⁻ cells for clarity. N = naïve. (B) Pleural cavity worm burden from mice in (A). (C) Pleural cavity cells elicited upon intra-thoracic injection of thioglycollate or (D), after infection with L. sigmodontis, using flow cytometric gating schemes shown in fig. S1, D and E. N = naïve. (E) Total pleural macrophages elicited by thioglycollate after i.v. injection of PBS, or CL-liposomes from 2 days prior (D -2) or on the day of (D 0) thioglycollate injection. (F) Total pleural macrophages and the proportion producing RELMα on day 10 post-L. sigmodontis infection after daily i.v injection of CL-liposomes or PBS from days 6-9. (G) Frequency of Gr-1⁻/Ly-6C⁻ CD115⁺ (B1) or Gr-1^int/Ly-6C^high CD115⁺ (B2) monocytes in CD11b⁺ blood leukocytes on day 3 and 10 post-L. sigmodontis infection after daily i.v injection of PBS or CL-liposomes from days 2-5. Values in italics represent the mean ± SEM of 8 mice. (A-G) All results are representative of 2-3 independent experiments. Graphs present mean ± SEM of 3-8 mice. (C and D) *P <0.05, **P <0.01 and ***P <0.001, as determined using Kruskal-Wallis test, with Dunn’s post-test comparing all groups. (E) ***P <0.001, as determined using one-way analysis of variance (ANOVA) with Tukey’s post-test for multiple comparisons.

Fig. 2

Type-2 inflammation drives in situ proliferation of tissue macrophages. (A) Flow-cytometric analysis of Ki67 expression or BrdU incorporation by pleural macrophages from naïve mice or 3 days post-thioglycollate (Thio) injection. BrdU was administered 3 hours prior to analysis. Gating schemes for Ki67 and BrdU analysis are shown in fig, S1B and 3A, respectively. Results are pooled data from 2 independent experiments with between 8 and 11 total mice per group. (B) As (A) but BrdU injection and analysis of cells was performed on day 10 post-infection with L. sigmodontis. Results are pooled data from 2 independent experiments with between 5 and 11 total mice per group, and are representative of at least 3 further independent experiments. (C) Total pleural macrophages and the proportion staining positive for BrdU and RELMα from naïve and day 10 L. sigmodontis-infected C57BL/6 (WT) and Il4^−/− (−/−) mice, as determined by flow cytometry. Data are representative of two independent experiments. Graphs present mean ± SEM of 7-8 mice per group. (A and B) **P <0.01 and ***P <0.001, as determined by 2-tailed t test. (C) *P <0.05, **P <0.01 and ***P <0.001, as determined using one-way ANOVA with Tukey’s post-test for multiple comparisons.

Fig. 3

Macrophage proliferation is an innate tissue hyperplasia triggered by IL-4. Mice were treated i.p. with PBS or IL-4c on days 0 and 2 and analyzed on day 4. (A) Composition of peritoneal cells, the proportion of peritoneal macrophages positive for BrdU, Ki67 or RELMα, and representative flow cytograms depicting all peritoneal cells. Graphs present mean ± SEM of 5 mice per group; representative of 10 independent experiments. (B) Total F4/80⁺ CD11b^low Kupffer cells, BrdU incorporation by Kupffer cells, and gating scheme depicting Kupffer cells and BrdU versus MHCII staining as determined by flow cytometry. Data shown are of livers pooled from 3 PBS-treated mice, or individual IL-4c-treated mice, and are combined from two independent experiments. (C) As (A) but in the pleural cavity. Graph presents mean ± SEM of 3 mice per group for cell composition, whereas BrdU incorporation is of pooled cells from 3 PBS-treated mice, or individual IL-4c-treated mice. Data are representative of 5 independent experiments. (D) Flow cytometric analysis of chimerism of blood monocytes and pleural macrophages and number of pleural macrophages in Cd45.1 congenic mice given CD45.2⁺ bone marrow cells after partial-body irradiation, and subsequently treated with PBS or IL-4c i.p., or intra-thoracic thioglycollate at week 8 post reconstitution. Representative CD45.1 and CD45.2 staining gated on CD11b⁺ CD115^high blood monocytes or F4/80⁺ pleural macrophages. Graphs depict mean ± SEM of 4 mice per group; representative of 2 independent experiments. (E) As (A) but in C57BL/6 (WT) and Rag1^−/− (−/−) mice. Graphs depict mean ± SEM of 3-4 mice per group; representative of 2 independent experiments.

Fig. 4

IL-4 triggers inflammatory macrophages to proliferate and alternatively activate. The proportion of peritoneal macrophages positive for BrdU, Ki67 or RELMα are shown, and representative flow cytograms depicting all peritoneal cells, from mice injected i.p. simultaneously with thioglycollate and PBS or IL-4c. IL-4c and PBS injections repeated on day 2 and analysis performed on day 4. Data are representative of two independent experiments, with 5 mice per group.