Type I and III IFNs produced by the nasal epithelia and dimmed inflammation are features of alpacas resolving MERS-CoV infection

Abstract

While MERS-CoV (Middle East respiratory syndrome Coronavirus) provokes a lethal disease in humans, camelids, the main virus reservoir, are asymptomatic carriers, suggesting a crucial role for innate immune responses in controlling the infection. Experimentally infected camelids clear infectious virus within one week and mount an effective adaptive immune response. Here, transcription of immune response genes was monitored in the respiratory tract of MERS-CoV infected alpacas. Concomitant to the peak of infection, occurring at 2 days post inoculation (dpi), type I and III interferons (IFNs) were maximally transcribed only in the nasal mucosa of alpacas, while interferon stimulated genes (ISGs) were induced along the whole respiratory tract. Simultaneous to mild focal infiltration of leukocytes in nasal mucosa and submucosa, upregulation of the anti-inflammatory cytokine IL10 and dampened transcription of pro-inflammatory genes under NF-κB control were observed. In the lung, early (1 dpi) transcription of chemokines (CCL2 and CCL3) correlated with a transient accumulation of mainly mononuclear leukocytes. A tight regulation of IFNs in lungs with expression of ISGs and controlled inflammatory responses, might contribute to virus clearance without causing tissue damage. Thus, the nasal mucosa, the main target of MERS-CoV in camelids, seems central in driving an efficient innate immune response based on triggering ISGs as well as the dual anti-inflammatory effects of type III IFNs and IL10.

Fig 1. Viral loads in nasal swabs and respiratory tissues of MERS-CoV-infected alpacas.

(A) Viral RNA (left) and infectious MERS-CoV (right) loads from nasal swab samples collected at the day of euthanasia. (B) Viral RNA from respiratory tissues of alpacas collected at different dpi. Viral loads were determined by the UpE real-time RT-qPCR (A and B). Each bar represents the mean Cq value +SD of infected tissues from 3 animals euthanized on 0, 1, 2, 3 and 4 dpi, respectively (in total 15 animals). Statistical significance was determined by Tukey’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Infectious MERS-CoV loads from respiratory tissues of alpacas at 2 dpi. Two out of three animals (2/3) displayed infectious virus in lungs. Dashed lines depict the detection limit of the assays. Cq, quantification cycle; TCID50, 50% tissue culture infective dose.

Fig 2. Histopathological changes and viral detection in respiratory tracts of alpacas inoculated with MERS-CoV.

All respiratory tissues were fixed in 10% neutral-buffered formalin. (A) Nasal turbinate tissue sections of alpacas from the non-infected group (0 dpi), and from those necropsied on1 to 4 dpi. (B) Tissue sections of trachea, bronchus and lung (bronchiole and alveoli) from infected alpacas at 2 dpi. Original magnification: ×400 for all tissues. See S1 Table for the detailed distribution of MERS-CoV antigen (N protein) in respiratory tracts. Abbreviations: H/E, hematoxylin and eosin stain; IHC, immunohistochemistry; LB, large bronchus; SB, small bronchus.

Fig 3. Kinetics of innate immune response genes induced at the nasal epithelia from MERS-CoV Qatar15/2015 infected alpacas.

(A) The nasal epithelium of each alpaca (AP1 to AP15) was micro-dissected, as assessed by IHC (see the image of the nasal turbinate section in the upper right). Infected (orange)/non-infected (green) areas were isolated by LCM for RNA extraction and conversion to cDNA. The Fluidigm Biomark microfluidic assay was used to quantify transcripts of innate immune genes at different dpi. After normalization, Fc values between controls and infected animals were calculated. The resulting heatmap shows color variations corresponding to log2 Fc values; blue for increased and red for decreased gene expression values of infected animals compared to control animals, respectively. IFNβ was normalized with 1 dpi samples because it was not detected at 0 dpi. Grey rectangles indicate no detection of the corresponding gene. TFs, transcription factors; CKs, chemokines; ADs, adaptors; RT, receptor. MERS-CoV+ and MERS-CoV-, MERS-CoV positive and negative epithelium areas as assessed by IHC, respectively. (B) Average Fc of IFNs, ISGs, PRRs, IRF7, IL10 and TRIM25 genes in MERS-CoV+ nasal epithelia (white bars) and MERS-CoV-nasal epithelia (black bars) as assessed by IHC. Data are shown as means of ±SD. Statistical significance was determined by Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001 when compared with the average values of non-infected alpacas (n = 3); #P < 0.05; ##P < 0.01; ###P < 0.001 when comparisons between groups are performed at different dpi.

Fig 4. Kinetics of alpaca innate immune responses at the nasal submucosa in response to MERS-CoV Qatar15/2015.

(A) The nasal submucosa of each alpaca (AP1, 2, 3, 7, 8, 9, 13, 14 and 15) was micro-dissected and areas underlying infected (orange)/non-infected (green) epithelium, as assessed by IHC (see the right panel), were selected and isolated for RNA extraction and conversion to cDNA. The Fluidigm Biomark microfluidic assay was used to quantify transcripts of innate immune genes at different dpi. After normalization, Fc values between controls and infected animals were calculated. The resulting heatmap shows color variations corresponding to log2 Fc values; blue for increased and red for decreased gene expression, respectively. The grey rectangles indicate no detection of the corresponding gene. TFs, transcription factors; CKs, chemokines; ADs, adaptors; RT, receptor. MERS-CoV+ and MERS-CoV-, MERS-CoV positive and negative epithelium areas as assessed by IHC, respectively. (B) Average Fc of ISGs, PRRs, IRF7, IL10, IL1β, IL8, NLRP3, CASP1and PYCARD genes in the submucosa underneath IHC MERS-CoV+ nasal epithelium (white bars) and IHC MERS-CoV- nasal epithelium (black bars). Data are shown as means of ±SD. Statistical significance was determined by Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001 when compared with the average values of non-infected alpacas (n = 3); #P < 0.05; ##P < 0.01; ###P < 0.001 when comparisons between groups are performed at different dpi.

Fig 5. Kinetics of alpaca innate immune responses at the tracheal level in response to MERS-CoV Qatar15/2015.

(A) Trachea samples were obtained by scraping MFPE sections from control (AP13-15) and infected alpacas (AP1-12), followed by RNA extraction and conversion to cDNA. A Fluidigm Biomark microfluidic assay was used to quantify transcripts of innate immune genes at different dpi. After normalization, Fc values between controls and infected animals were calculated. The resulting heatmap shows color variations corresponding to log2 Fc values; blue for increased and red for decreased gene expression, respectively. The grey rectangles indicate no detection of the corresponding gene. TFs, transcription factors; CKs, chemokines; ADs, adaptors; RT, receptor. (B) Average fold changes of ISGs, PRRs, IL8, IL1β and IRF7 genes in trachea. Data are shown as means of ±SD. Statistical significance was determined by Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001 (n = 3) when compared with non-infected alpacas (n = 3). (C) On 2 dpi, no inflammatory cells were observed in the tracheal submucosa of AP4 (see H/E stained MFPE tracheal section at the left). The graph at the right depicts Fc of mRNA transcripts for CCL2 and CCL3 in relation to control animals. (D) Infiltration of monocytes and lymphocytes in the tracheal submucosa of AP6 (see H/E stained MFPE tracheal section at the left) was associated with increased mRNA transcripts of CCL2 and CCL3 (see the bar graph at the right).

Fig 6. Kinetics of alpaca innate immune responses in the lungs following MERS-CoV Qatar15/2015 infection.

(A) The lung sample of each infected alpaca (AP1 to AP15) was isolated for RNA extraction and conversion to cDNA. A Fluidigm Biomark microfluidic assay was used to quantify transcripts of innate immune genes at different dpi. After normalization, Fc values between controls and infected animals were calculated. The resulting heatmap shows color variations corresponding to log2 Fc values; blue for increased and red for decreased gene expression, respectively. The grey rectangles indicate no expression of the corresponding gene. TFs, transcription factors; CKs, chemokines; ADs, adaptors; RT, receptor. (B) Bar graphs representing average mRNA fold changes of ISGs (ISG15, MX1, CXCL10 and OAS1), IRF7, NLRP3, the inflammatory cytokines IL1β, TNFα, IL10 and IL15 genes in lung. Data are shown as means of ±SD. Statistical significance was determined by Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001 (n = 3) compared with non-infected alpacas (n = 3); #P < 0.05; ##P < 0.01; ###P < 0.001 (n = 3) when comparisons between groups are performed at different dpi.

Fig 7. Infiltration of leukocytes in alveoli are correlated with upregulation of CCL2 and CCL3 mRNA.

(A) Relative expression of CCL2 and CCL3 mRNA (Bar graphs at the left) was performed with a microfluidic PCR assay in the lung of alpacas (except AP6, see text). Leukocytes were counted in 3 microscopic fields (400X) per lung section in all animals (except AP6), including non-infected controls. Relative expression levels of CCL2 and CCL3 were plotted against the number of inflammatory cells (dot plot at the right). Correlation coefficients were established using the Spearman’s correlation test (right panel). (B) The number of leukocytes in alveoli was the highest in AP5, lower in AP10 (arrows), occasional in AP7 (arrow) and hardly detectable in AP13. Original magnification: ×1000 for all samples.

Fig 8. Tentative mechanistic model for MERS-CoV-induced protective innate immune responses in alpaca respiratory tracts.

(A) Upon MERS-CoV infection of nasal epithelial cells, PRRs and IRFs are engaged to induce IFNs and ISGs exerting their effects in an autocrine or paracrine manner. Upregulation of IL10 combined with the action of type III IFNs will result in a dimmed synthesis of proinflammatory cytokines under NF-κB control. Induced chemokines lead to focal, mild infiltration of leukocytes. The paracrine effect of IFNs is evidenced in the nasal submucosa where ISGs are upregulated without endogenous induction of IFNs. IL10 and type III IFNs will act on the NF-κB pathway preventing production of IL8 and IL1β mRNAs depriving the NLRP3 inflammasome of its substrate. Slight increased levels of IRF5 mRNAs will indicate the presence of few M1 macrophages in the submucosa. Blue, red and white arrows indicate upregulation, downregulation and unaltered gene transcription respectively. (B) Concomitant to decreased viral loads, transcription of ISGs is lowered in trachea and lungs where IFNs are not induced. Infiltration of mainly mononuclear leukocytes occurs in the lungs as a result of chemokine synthesis in the absence of IRF5 induction but upregulation of IL15. Thus, reduced number of M1 macrophages and activation of NK cells will contribute to a controlled inflammation and clearance of the virus. PI, Pro-inflammatory cytokines; NI, not induced.