The RNA uridyltransferase Zcchc6 is expressed in macrophages and impacts innate immune responses

<div><p>Alveolar macrophages orchestrate pulmonary innate immunity and are essential for early immune surveillance and clearance of microorganisms in the airways. Inflammatory signaling must be sufficiently robust to promote host defense but limited enough to prevent excessive tissue injury. Macrophages in the lungs utilize multiple transcriptional and post-transcriptional mechanisms of inflammatory gene expression to delicately balance the elaboration of immune mediators. RNA terminal uridyltransferases (TUTs), including the closely homologous family members Zcchc6 (TUT7) and Zcchc11 (TUT4), have been implicated in the post-transcriptional regulation of inflammation from studies conducted <i>in vitro</i>. <i>In vivo</i>, we observed that Zcchc6 is expressed in mouse and human primary macrophages. Zcchc6-deficient mice are viable and born in Mendelian ratios and do not exhibit an observable spontaneous phenotype under basal conditions. Following an intratracheal challenge with <i>S</i>. <i>pneumoniae</i>, Zcchc6 deficiency led to a modest but significant increase in the expression of select cytokines including IL-6, CXCL1, and CXCL5. These findings were recapitulated <i>in vitro</i> whereby Zcchc6-deficient macrophages exhibited similar increases in cytokine expression due to bacterial stimulation. Although loss of Zcchc6 also led to increased neutrophil emigration to the airways during pneumonia, these responses were not sufficient to impact host defense against infection.</p></div

Zcchc6 minimally directs neutrophil emigration during pneumococcal pneumonia.

<p>(A) Total BAL cell counts (B) alveolar macrophage numbers and (C) neutrophil emigration were assessed by bronchoalveolar lavage from <i>Zcchc6</i>^<i>+/+</i> or <i>Zcchc6</i>^<i>-/-</i> mice infected with of Sp19 i.t. for 4 hours. **p<0.01 by Student’s t-test. (D) Bacterial lung burdens were determined from <i>Zcchc6</i>^<i>+/+</i> or <i>Zcchc6</i>^<i>-/-</i> mice 30 hours post intratracheal instillation of Sp19. (E) Cytokine mRNA expression was measured by qRT-PCR on total lung left lobe RNA isolated from Sp19-infected <i>Zcchc6</i>^<i>+/+</i> and <i>Zcchc6</i>^<i>-/-</i> mice at 4 hours post infection. Data are normalized to 18S rRNA levels and expressed as fold induction over <i>Zcchc6</i>^<i>+/+</i>. n = 6,8; *p<0.05 by Student’s t-test. (F) CXCL1 protein levels were measured by ELISA in supernatants collected from cultured BMDMs (n = 4,3) stimulated with 1 x 10⁶ CFU/mL Sp19.</p

Establishment of a mouse model of Zcchc6 deficiency.

<p>(A) Gene rearrangement in Zcchc6^-/- mice at the DNA level was confirmed by PCR of tail genomic DNA. (B) Zcchc6 mRNA expression was measured by qRT-PCR from total left lobe lung RNA and normalized to 18S rRNA. Data expressed as mean and SEM, ***p<0.001 by Student’s t-test (C) Immunoblot analysis of Zcchc6 protein expression across multiple tissue cell lysates. (D) Genotypes of pups weaned from Zcchc6^+/- x Zcchc6^+/- crosses. Dotted line indicated expected frequency of homozygous animals.</p

Zcchc6 and Zcchc11 expression in adult mouse tissues.

<p>(A) Total RNA was prepared from C57BL/6 mouse tissues and was assayed for Zcchc6 and Zcchc11 mRNA expression by qRT-PCR. Data are normalized to 18S rRNA and displayed as fold induction over Zcchc11 expression (n = 3,3). *p<0.05 ***p<0.001 by two-way ANOVA with Bonferroni post-test. (B)(C) Zcchc6 protein expression was measured by immunoblot in primary human monocyte derived macrophages (4 donors), human monocytes (5 donors), and human alveolar macrophages (3 donors). Lung lysates generated from WT or Z6 deficient mouse lungs were used as a control in (C). Zcchc6 protein expression was assessed in (D) PMA or vehicle treated U937 or THP-1 human monocytic cell lines and in (E) mouse bone marrow-derived macrophages during a 7-day time-course of differentiation.</p

Top 20 GO Biological process terms.

<p>Top 20 GO Biological process terms.</p

Homeostatic macrophage cell numbers are unchanged in Zcchc6-deficient mice under basal conditions.

<p>(A) Total alveolar macrophages and (B) airspace neutrophils were isolated by bronchoalveolar lavage and quantified by hemocytometer and cellular differential analysis by Diffquick staining. (C) Pleural macrophages were collected by lavage and quantified by hemocytometer. Data were determined to be statistically non-significant by Student’s t-test.</p