CCR2 defines a distinct population of NK cells and mediates their migration during influenza virus infection in mice

Abstract

Natural killer (NK) cells are innate lymphocytes that play an important role in control of viral infections. We recently showed that intranasal infection of mice with influenza virus induced the accumulation of NK cells in the airways. NK cells however did not proliferate in the airways or in the draining lymph node, but in the bone marrow mainly. As also monocyte-precursors undergo vigorous proliferation in the bone marrow (BM) during infections and then egress CCR2-dependently, we decided to determine the role of CCR2 in NK cell migration during intranasal influenza virus infection. We show that a unique population of NK cells in the BM expressed CCR2 and that monocyte chemotactic protein-1 (MCP-1), one of the CCR2 ligands, was produced in the airways of influenza virus infected mice. Analysis of BM chimeric mice reconstituted with a mix of wild-type (wt) and CCR2-deficient BM cells showed that upon influenza virus infection, a significantly lower proportion of CCR2-deficient than wt NK cells was recovered from the bronchoalveolar lavage (BAL). Taken together, our data demonstrate that during influenza virus infection a proportion of NK cells migrate in a CCR2-dependent fashion.

Figure 1. A subset of NK cells expresses the CCR2 receptor.

(A) Gating strategy (upper panel) for NK cells (TCRβ⁻NK1.1⁺DX5⁺) and representative histograms (lower panel) showing CCR2 expression on NK cells recovered from the indicated organs (blood, lung, BM and spleen) of naïve C57BL/6 (wt) or CCR2^−/− mice. (B,C) Expression of CD27 and CD11b (C) or KLRG1 on electronically gated NK cells (TCRβ⁻NK1.1⁺) or CCR2⁺ NK cells. The depicted FACS plots are representatives of 9 mice that were analyzed in two independent experiments. Statistical analysis was performed using a Mann-Whitney U test (C) and KLRG-1-expression on CCR2⁺ or CCR2⁻ NK cells is not significantly different (Mann-Whitney U test, *, P<0.05).

Figure 2. MCP-1 is expressed in the airways of influenza virus infected mice.

(A) C57BL/6 mice were infected with HKx31 and the presence of MCP-1 in the BALF was determined by ELISA at the indicated days post-infection. Data shown are means+S.E.M. from one experiment with 5 mice per time point. Similar results were found in an independent experiment at day 2 and 5 post-infection.

Figure 3. NK cells migrate CCR2-dependent to the BAL during influenza virus infection.

Mixed BM chimeric mice were constructed by injecting a mix of BM from CCR2^−/− (CD45.2) and C57BL/6.SJL (CD45.1) into lethally irradiated CD45.1.2. recipients. After 6–8 weeks, mice were infected with influenza virus or left uninfected. (A) Representative FACS plots showing CD45.1 and CD45.2 staining of NK cells (TCRβ⁻NK1.1⁺) recovered from the indicated organs 5 days p.i. (B-D) Ratio of CD45.2 (CCR2^−/−)/CD45.1 (wt) NK cells calculated by dividing absolute numbers of CD45.2⁺ NK cells by absolute numbers of CD45.1⁺ (wt) NK cells. Ratio of CD45.2/CD45.1 NK cells recovered from the indicated organs shown as average ± S.E.M. at the indicated days (B) and shown as individual mice at day 5 (C), and the ratio in the spleen and BM of individual mice are connected by a line at day 5 (D) after influenza virus infection. Representative results of two independent experiments are shown with 4–6 mice per group. Statistical analysis was performed using a Mann-Whitney U test. *, P<0.05.