CD11a KO, CD11b KO, CD11d KO, CD45.1 and CD45.2 wild type mice were purchased from the Jackson laboratory. CD11c KO mice were kindly given by Dr. Christine Ballantyne (Baylor University), as we previously described in our publication (8–10). Intercellular adhesion molecule-1 (ICAM-1) KO mice were kindly given by Dr. Gregory Priebe (Boston Children’s Hospital) (11). They were housed under specific pathogen free condition, with 12-hour light and dark cycles. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Boston Children’s Hospital.

Whole bone marrow cells were isolated from mice and stained on ice with various antibody cocktails to identify HSPC compartment. As we previously described (9), the lineage marker includes anti-mCD11b (M1/70), anti-mGr1 (RB6-8C5), anti-mCD3 (145-2C11), anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-mB220 (RA3-6B2), anti-mNK1.1 (PK136), and anti-mTer119 (TER-119). The other mouse antibodies include anti-mCD45.1 (A20), anti-CD45.2 (104), anti-mIgM (R6-60.2), anti-mC-kit (2B8), anti-mSca-1 (D7), anti-mCD34 (RAM34), anti-mCD150 (TC15-12F12.2), anti-mCD48 (HM48-1), anti-mIL7Rα (A7R34), and anti-mFcR (93), and its isotype control hamster IgG (A19-3). Antibodies were purchased from Biolegend (San Diego, CA) and BD Biosciences (Franklin Lakes, NJ). Cell counting was done by applying Sphero AccuCount beads (ACBP-50; Spherotech Inc, Lake Forest, IL). Data were acquired on a Canto II cytometer (BD Biosciences) and analyzed using FlowJo software (flowJo LLC, Ashland, OR).

Cell proliferation was examined either with Ki67 staining or 5-bromo-2’-deoxyruridine (BrdU) staining. For Ki67, cells were subjected to intracellular staining using anti-mKi67 antibody (11F6, Biolegend). For BrdU incorporation, mice were injected intraperitoneally (i.p.) with 1 mg of BrdU. To detect BrdU incorporation into bone marrow hematopoietic cells, BD cytofix/cytoper Plus kit (555028, BD Biosciences) and Alexa fluor 488-conjugated anti-BrdU antibody (3D4, Biolegend) were applied according to the manufacturer’s protocol. Then cells were subjected to flow cytometry analysis.

Mice were subjected to lipopolysaccharide (LPS, from Pseudomonas aeruginosa 10, 10 mg/kg; Sigma, St. Louis, MO) injection via tail vein. Mice were euthanized at indicated time points for analysis.

To generate mixed bone marrow chimeras, recipient mice on the C57BL/6 background were irradiated with 2 doses of 550 rad with 4-hour intervals. Wild-type (WT, CD45.1) and WT (CD45.2) derived bone marrow cells at WT (CD45.1) and CD11a KO (CD45.2) derived bone marrow cells were mixed at the ratio of 1:1 with total of 5 x 10⁶ cells and intravenously (i.v.) injected into the tail vein of lethally irradiated recipients. Mice were evaluated for the reconstitution of the immune compartment at various time points after bone marrow transplantation. To prevent bacterial infection, the mice were provided with autoclaved drinking water containing sulfatrim for 1 week prior to and for 4 weeks after irradiation.

Plasma IL-27 level or IL-27 levels in cell culture supernatant were measured using IL-27 ELISA kit per the company’s protocol (Thermo Fischer). TNF-α, KC, IL-1β, IL-6, IL-10, IL-12p70, and IFNγ in plasma or cell culture supernatant were measured using mouse cytokine 7-plex (Mesoscale, Rockville, MD) per the company protocol.

Bone marrow cells were isolated and red blood cells were lyzed. Single cell suspension was cultured in DMEM medium containing 10% FCS and recombinant murine M-CSF (rmM-CSF, Peprotech, 100 unit/ml) for 3 days. After equal volume of fresh medium containing 10% FCS and rmM-CSF (100 unit/ml) was added, cells were cultured for additional 4 days. At the end of differentiation, supernatant and non-adherent cells were discarded. The adherent cells were deemed as bone marrow-derived macrophage (BMDM) and used for further experiments. Regarding LPS stimulation, BMDMs were cultured with medium or LPS (1 µg/ml) for 24 hours. In some experiment, we used CD11a inhibitor GSK2344307A and GSK2344343A (10 µM) (12).

Data were analyzed as indicated in the figure legend. Statistical significance was defined as P< 0.05. All the statistical calculations were performed using PRISM5 software (GraphdPad Software, La Jolla, CA).

Because CD11a is known to be expressed ubiquitously on peripheral leukocytes, we checked to see if CD11a was also expressed on hematopoietic stem and progenitor cells (HSPCs). We found that CD11a was expressed on all of long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs), multipotent progenitors (MPPs), common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) ( Figure 1A ). The number of lineage⁻sca-1⁺c-kit⁺ (LSK) cells and CMPs was comparable between WT and CD11a KO mice ( Figure 1B ). No difference in proliferation was observed ( Figure 1C ).

To understand how the bone marrow (BM) cells respond to infectious stimuli, we performed LPS stimulation in vivo. LSK number significantly increased in CD11a KO mice along with higher proliferation based on Ki67 expression ( Figure 1D ). Only 4.5% of WT LSK cells were Ki67 positive, while around 8% of CD11a KO LSK showed Ki67 expression. In contrast to LSK, CMP number in WT and CD11a KO mice upon LPS injection did not differ ( Figure 1E ). CMP proliferation was much higher than LSK in both WT and CD11a KO mice based on Ki67 expression. In WT mice, 50% of CMP expressed Ki67, while CD11a KO CMPs expressed even slightly more Ki67 than WT counterpart. This explains higher neutrophil counts in CD11a KO mice ( Figure 1F ).

ICAM-1 is a major ligand for CD11a. Thus, we also examined if ICAM-1 KO mice would behave similarly to CD11a KO mice. However, there was no difference in LSK and CMP number between WT and ICAM-1 KO mice following LPS stimulation ( Figure 1G ). Previously, we also examined the role of other β2 integrins CD11b, CD11c and CD11d in HPSCs using CD11b KO, CD11c KO and CD11d KO mice. We found that CD11cKO mice had significantly less LSK cells, but CD11bKO or CD11dKO mice did not have any difference in LSK number compared to WT mice following LPS injection (8). Thus, the expanded LSKs in CD11aKO mice upon LPS stimulation suggests a distinctive biological role of CD11a among β2 integrins.

To investigate whether higher CD11a KO LSK proliferation was intrinsically driven by CD11a, we created mixed chimeric mice by injecting the mixture of bone marrow cells at the ratio of 1 :1 into lethally irradiated recipients. Six weeks later, we monitored the peripheral blood. There was no statistical significance between CD45.1 and CD45.2 cells in both chimeric mice ( Figure 2A ). We then examined their bone marrow cells. There was no statistically significant difference in CD45.1 and CD45.2 derived LSK and CMP ( Figure 2B ). Proliferation was also not different between genotypes ( Figure 2C ).

We then examined in vivo LPS stimulation in the chimera. We did not find any statistically significant difference in the ratio between CD45.1 and CD45.2 components in LSK and CMP ( Figure 3A ). Furthermore, we did not find any difference in proliferation between genotypes ( Figure 3B ). This result points out that higher proliferation in CD11a KO LSK in CD11a KO mice upon LPS injection was not intrinsically driven. This result was in line with our finding that ICAM-1 KO mice did not show similar phenotype with CD11a KO mice as above.

Previously, mediators that drive emergency granulopoiesis have been extensively examined in the setting of LPS stimulation, among which IL-27 was shown to expand LSK population (13). Thus, we hypothesized that CD11a KO mice might possess higher IL-27 levels after LPS stimulation. As hypothesized, IL-27 was significantly higher in plasma in CD11a KO mice after LPS stimulation ( Figure 4A ). Plasma from CD11b KO and CD11c KO mice showed a trend that their IL-27 levels were higher than that of WT mice, though statistically not signifincat ( Figure 4A ). IL-27 is primarily produced by myeloid cells (14–16). Therefore, we tested if CD11a KO macrophages produced higher levels of IL-27 in vitro, in which case BM derived macrophage (BMDM) was differentiated and used. In line with the finding in vivo, we observed that IL-27 level was higher in CD11a KO BMDM stimulated with LPS compared to WT BMDM ( Figure 4B ). We also tested CD11a inhibitors GSK2344307A and GSK2344343A in WT BMDM in vitro. These inhibitors directly bind to the ligand binding domain called the I domain in CD11a to block CD11a-ligand binding (12). Similar with CD11a deficiency, both CD11a inhibitory compounds enhanced IL-27 production stimulated by LPS as well ( Figure 4C ). To understand other cytokine profiles, we also measured TNF-α, KC, IL-1β, IL-6, IL-10, IL-12p70 and IFNγ. We found that only IL-10, was significantly elevated in CD11a KO both in vivo but not in CD11b, CD11c, and CD11d KO mice ( Figure 4D ). We also found that IL-10 level was higher in CD11a KO macrophages compared to WT macrophages in vitro ( Figure 4E ).