We thank Dr. Carson Harrod for critical reading of the manuscript. We also thank Mr. Minghui Zeng and Drs. Mahesh Kathania and Prashant Khare for their contributions. This work was supported by grants from the American Cancer Society (Research Scholar grant, 122713-RSG-12-260-01-LIB) and the Sammons Cancer Center (Pilot Project grant) to K. Venuprasad.

모든 실험은 베일러 연구소의 기관 동물 관리 및 사용위원회 (IACUC)의 지침에 따라 수행되었다. 1. 배양 비 typeable 헤모필루스 인플루엔자 (NTHi)과 접종 준비 <ol><li> 초콜릿 한천 플레이트 접시 NTHi를 37 ° C, 5 % CO 2에서 하룻밤 가습 CO 2 배양기에서 거꾸로 판을 유지한다. 다음 날, 문화 뇌 심장 주입 국물에 박테리아. 그런 다음, 멸균 1.5 ML 튜브로 국물 1 ㎖를 추가합니다. </li><li> 실온에서 5 분 동안 4000 × g으로 원심 분리기. 그 다음, 상층 액을 제거하고 멸균 1X PBS 1 ㎖에 펠릿을 다시 일시. 일단이 단계를 반복합니다. </li><li> 분광 광도계로 파장 600nm에서의 희석 배양 흡광도 1X PBS 900 μL에 재현 탁 용액 100 μl를 전송하고 측정한다. </li><li> 생존 및 집락 형성 단위를 결정초콜렛 아가 플레이트에 1시 10분 연속 희석을 배양 CO 2, 37 ℃에서 밤새 배양하고 5 % 접종 S (CFUs). </li><li> 이전 단계에서 결정된 가능성에 기초하고 CFUs, 20 × 106 CFU / ml의 최종 농도로 접종을 조정한다. </li></ol> 2. 인트라 기관 (IT) 점적 <ol><li> 150 ㎎ / ㎖ + 케타민 20 ㎎ / ㎖ 용액과 자일 라진 마우스 0.1 ml / 10 g 체중으로 마우스의 복강 (IP)을 주입한다. 절차는 동물을 따뜻하게 유지하는 광원과 후드에서 수행해야합니다. </li><li> 마우스가 적절하게 마취되었는지 확인하기 위해 마우스의 인터 공간을 꼬집어. </li><li> 그 뒷면에 마우스를 놓습니다. 목 복부 영역을 면도 클로르헥시딘, 70 % 에틸 알코올로 소독. </li><li> 쥐 치아 집게를 사용 넥 부의 상부 복부 피부를 리프트. 메스를 이용하여 흉선 상기 작은 절개 (0.5-1.0 cm)를 만들기조심스럽게 기관을 노출 근육을 해부하다. <ol><li> 27 G 바늘 1 mL를 주사기를 사용하여 서서히 다른 0.05 ㎖의 공기 다음 컨트롤과 같은 제 1 염에서 얻어진 용액 0.05 NTHi 다음 0.05 ml의 공기를, 주사. 약 1 분 동안 수직 동물을 유지합니다. </li></ol></li><li> 수술 접착제와 절개를 닫습니다. 약 10 분 동안 옆으로 누운 따뜻한 마우스를 유지합니다. </li></ol> 3. BALF 컬렉션 <ol><li> 감염 후 자궁 전위 24 시간으로 마우스를 희생. </li><li> 해부 가위와 마우스의 복강을 열고 출혈 복부 대동맥을 잘라. 폐의 복부 부분을 노출. </li><li> 기관을 노출하고 20 게이지 카테터를 삽관. BALF 버퍼의 0.8 ㎖ (1X PBS, 0.1 % 덱 스트로스, 10 U / ㎖ 헤파린)를 주입하고 부드러운 흡입에 의한 세척를 수집합니다. 이 과정을 세 번 반복합니다. </li><li> hemocyto BALF를 사용하여 현탁액 100 ㎕의 세포의 총 수를 카운트트리 판 블루 염색으로 10 배 배율 아래 미터. </li><li> 수정 김사 염색 슬라이드를 준비합니다. 5 분 500 XG에 cytospin 원심 분리기의 적절한 우물에 폐포 세척액의 나누어지는 100 μL. 10 분 동안 슬라이드를 건조 및 제조 업체의 프로토콜에 따라 수정 김사 염색을 사용하여 세포를 얼룩. </li><li> 이러한 개화 (FACS) 정렬 활성화 세포, 공 초점 현미경, 유전자 발현 및 웨스턴 블롯 11-13 후속 분석을 위해 남아있는 세포를 사용합니다. </li></ol> 4. 조직 병리학 준비 <ol><li> 단계 2.4에 설명 된대로 조직 병리학 분석을 위해, NTHi와 마우스를 감염. 감염 24 시간 후 자궁 전위에 의해 동물을 희생. 폐와 앞서 언급 한 바와 같이 기관을 노출 해부 가위로 흉강을 열고 잘라. 그리고, 기관에 20 게이지 카테터를 삽입합니다. </li><li> 4 % 포르말린에서 제조 된 2 % 따뜻한 아가로 오스 용액으로 폐를 입력합니다. 일단폐는 완전히 아가로 오스 솔루션을 응고 폐의 상단에 장소 얼음, 팽창된다. </li><li> 그런 다음, 조심스럽게 흉부 플러그를 제거하고 헤 마톡 실린 및 에오신 (H &amp; E) 부분에 대한 처리 될 때까지 1X PBS에서 4 % 포르말린 50 ㎖ 용액에 넣어. </li></ol> BALF에서 세포를 염색 5. FACS <ol><li> 96 웰 V 바닥 마이크로 타이 터 플레이트에 BALF 유체 1 × 10 5 세포를 넣습니다. </li><li> 4 ° C에서 3 분 동안 4,000 XG에 300 차가운 1X PBS의 μL 및 스핀 세포를 씻으십시오. </li><li> (1 : 1000 희석) 1X PBS 100 ㎕를 세포 염색 용액으로 세포를 염색하고 15 분 동안 실온에서 유지한다. </li><li> 4 ° C에서 3 분 동안 4,000 XG에 차가운 1X PBS 300 μL와 세포를 씻으십시오. </li><li> 하나를 포함 FACS 세척 완충액 [세척 완충액 (1X PBS, 2 % FBS, 1 mM의 EDTA, 0.1 % 나트륨 아 지드] 100 ㎕에 세포를 유지 : 100 희석 된 Fc 블록을 4 ℃에서 15 분 동안. </li><li> 4,000 XG에 원심 분리기 세포 3 분 및 저녁을 먹다 폐기ernatant. </li><li> 4 ℃에서 30 분 동안 FACS 세척 버퍼 (100 희석 1) 100 ㎕의 표면 얼룩 칵테일 얼룩 세포. </li><li> 4 ° C에서 3 분 동안 4,000 XG에 FACS 세척 버퍼 300 μL로 세 번 세포를 씻으십시오. </li><li> 실온에서 15 분 동안 4 % 파라 포름 알데하이드 100 ㎕에 세포를 고정한다. </li><li> 4 ° C에서 3 분 동안 400 XG에서 PBS 1 배 300 μL로 두 번 세포를 씻고 FACS 세척 버퍼 300 μL에 다시 일시 중지합니다. </li><li> 비드 유동 세포 계측법을 사용하여 단색 오염 제어를 만든다. <ol><li> 96 웰 V 바닥 마이크로 리터 판에 구슬 한 방울을 추가하고 PBS 1 배 200 μL 세척. </li><li> PBS와 구슬 1 배 100 μL에 염색 항체의 1 μl를 추가하고 5 분 동안 실온에서 품어. </li><li> PBS 1 배 300 μL로 두 번 구슬을 씻으십시오. </li></ol></li><li> 14 유동 세포 계측법에 의해 단일 염색 컨트롤과 세포를 분석 할 수 있습니다. </li></ol> RNA를 분리하기 위해 폐 조직의 균질화 6. <ol><li> 감염 후 폐 (20 ~ 40 mg)의 작은 조각을 수집하고 밤새 4 ° C에서 시약을 안정화 RNA에 보관합니다. 그리고, 다음 단계까지 -80 ℃에서 보관. </li><li> RNA 안정화 시약 없애 차가운 1X PBS의 폐 조직을 씻으십시오. </li><li> 1.5 ㎖의 원심 분리기 튜브 포함 용해 버퍼 (1 ml의 용해 버퍼 + 10 μL의 β-머 캅토 에탄올)로 조직을 놓습니다. </li><li> 뾰족한 가위를 사용하여 작은 조각으로 조직을 잘라 및 20 ~ 40 초 동안 무선 모터 펠렛 유봉을 사용하여 균질화. </li><li> 5 분 동안 얼음에 해물을 유지하고 단계 7.1를 진행합니다. </li></ol> 폐 7. RNA 분리 <ol><li> 3 분 동안 18,000 XG에 해물을 원심 분리기. 피펫으로 상층 액을 제거하고 조심스럽게 새로운 1.5 ML 튜브로 전송할 수 있습니다. </li><li> 해물을 70 % 에탄올 1 볼륨을 추가합니다. 피펫 즉시 혼합하고 2-4 분 동안 기다립니다. </li><li> 배치 스핀 컬럼에 해물의 700 μL까지 이동2 ml의 포집 관에서. </li><li> 15 초 동안 9,600 XG에 원심 분리기와 흐름을 통해 폐기하십시오. </li><li> 15 초 동안 9,600 XG에 350 μL에게 엄격한 세척 버퍼와 원심 분리기를 추가합니다. 흐름을 통해 폐기하십시오. </li><li> I 직접 스핀 컬럼 막에 80 μL의 DNase를 추가하고 실온에서 15 분 동안 유지. </li><li> 15 초 동안 9,600 XG에 스핀 컬럼 및 원심 분리기 350 μl의 엄격한 세척 버퍼를 추가합니다. 흐름을 통해 폐기하십시오. </li><li> 15 초 동안 9,600 XG에 500 ㎕의 RNA 세척 버퍼와 원심 분리기를 추가합니다. 흐름을 통해 폐기하십시오. </li><li> 2 분 동안 9,600 XG에 500 ㎕의 RNA 세척 버퍼와 스핀을 추가합니다. 흐름을 통해 폐기하십시오. </li><li> 1 분 동안 9,600 XG에 새로운 2 ㎖의 수집 튜브와 원심 분리기에 스핀 열을 놓습니다. 흐름을 통해 폐기하십시오. </li><li> 1 분 동안 9,600 XG에 스핀 컬럼 및 원심 분리기에 직접 30 ~ 50 μL의 RNase없는 물을 추가합니다. -80 ° C에서 보관 RNA. </li><li> RNAseq 분석 (14)을 수행합니다. </li></ol>

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The authors have nothing to disclose.

여기서, 우리는 박테리아 폐 병원체와 마우스의 폐를 접종 독특하고 최소 침습 절차를 설명합니다. 우리는이 절차 염증성 신호 전달 경로의 유전자 결핍 마우스를 이용하여 다양한 유전자의 기능을 연구하는데 사용될 수 있다는 것을 보여준다. 이 절차는 또한 바이러스 및 진균 감염에 폐 염증 반응을 연구하는데 사용될 수있다. 이러한 비내 에어로졸 또는 흡입 같은 다른 방법을 통해이 절차의 이점 (1)이 절차에서 병원성 접종 직접 하부 호흡기 점적되며 (2) 균액의 크기를 제어 할 수있는 기능; (3) 박테리아 핸들러 노출 위험을 감소시켰다. 수술 박테리아 주입 용 기관의 노출 치료가 주위 조직, 특히 혈관 손상을주지 않도록주의해야 중요한 단계이다. 기관 쉬에 박테리아의 점안에 의한 질식으로 마우스의 사망 가능성을 방지하기 위해주의 깊게 수행 할 수 울드. 접종 (50 μL)을 천천히 출시 및 공기 전과 접종 후 주입하는 것이 좋습니다. 잠시 동안 점안 다음 마우스가 수직 유지하고는 옆으로 누운 유지하는 빠른 복구를 용이하게한다. 이 방법의 주요 단점은 짧은 시간 내에 다수의 노출로 인해 수술의 필요에 불가능하다는 것이다. 잠재적 인 문제는 카테터 기관지 너무 깊게 배치하면, BALF 세포 수가 낮은 것 때문이다. 이 문제는 약간 위쪽으로 튜브를 당겨 해결 될 수있다. 기도 염증 질환의 발병률이 질환의 병태 생리에 대한 이해 전세계 3,16 증가되므로 가장 중요하다. 이 연구에 기술 된 기술은 조절 키 경로를 식별하는 데 도움이 될 수 있고, 개발을 촉진 할 수새로운 치료 방법의.

염증은 감염 인자에 대한 방어의 기본 면역 메커니즘입니다. 그것은 병원균 퇴치 및 손상된 조직의 복구를 촉진한다. 또한, 일반 건강 상태 1로 회복을 용이하게한다. 그러나, 조절 이상 염증은 종종 만성 염증성 질환이 리드. 기도 염증은 만성 폐쇄성 폐 질환 (COPD), 천식 및 폐 섬유증과 같은 3 가지 폐 질환의 초기 트리거이다. 비 typeable (봉입) 헤모필루스 인플루엔자 (NTHi)의 상부 및 하부 만성 폐 염증성 질환 4,5-와 연관된다. 그것은 만성 폐쇄성 폐 질환 4,6,7 어린이와 성인의 하부기도에서 분리 지배적 인 종이다. NTHi 감염 후 염증 반응은 (예컨대 TNF 및 IL-1β와 같은) 염증성 사이토킨의 상향 조절이 특징이며, B를 매개y를 미토 겐 활성화 단백질 키나아제 (MAPK) 및 수신자와 같은 수용체를 통해 핵 인자 κB (NF-κB) (TLR에) 8. 마우스 모델 때문에 서로 다른 유전자가 결핍 된 라인의 가용성의 폐 염증성 질환의 기본 병리학을 분석하는 매우 유용한 도구입니다. 여러 방법은 비내 점적 및 에어로졸 흡입 9,10- 포함한 실시간 / 감쇠 박테리아와 세균 제품을 접종하는데 사용되었다. 여기, 우리는 내 기관 점안을 보여줍니다. 덜 빈번하게 사용되지만,이 방법은 더욱 효율적이고 때문에 하부 호흡기 접종원 직접 전달 재현성이다.

<table border="0" cellpadding="0" cellspacing="0" width="705">
	<tbody>
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			<td height="21" style="height:21px;width:293px;">Chocolate agar plate</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">CAS50-99-7</td>
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			<td height="21" style="height:21px;width:293px;">Dextrose Anhydrous</td>
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			<td></td>
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			<td height="21" style="height:21px;width:293px;">Heparin</td>
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			<td height="21" style="height:21px;width:293px;">Deft quick solution</td>
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			<td>GS500-500ML</td>
			<td></td>
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			<td height="42" style="height:42px;width:293px;">Syringe needle 20/26G&#160;</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">(REF305115/175)</td>
			<td></td>
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			<td height="21" style="height:21px;width:293px;">Iml syringe</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">REF 309602</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Catheter 20GA</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">REF 381433</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Scissors, straight, 10 cm long</td>
			<td>kentscientific</td>
			<td>INS600393</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Iris Forceps, serrated, 10cm long</td>
			<td>kentscientific</td>
			<td>INS650915</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Tweezer #5 Stainless steel, 11cm long</td>
			<td>kentscientific</td>
			<td>INS600095</td>
			<td></td>
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			<td height="21" style="height:21px;width:293px;">10% Formalin</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:119px;">CAS 67-56-1</td>
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			<td style="width:119px;">35-1020</td>
			<td></td>
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			<td height="21" style="height:21px;width:293px;">5ml polystyrene round-bottom tubes</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">REF 352058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">1.5 ml Microcentrifuge tubes</td>
			<td style="width:127px;">Light Labs</td>
			<td style="width:119px;">A-7001-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Reasy Mini&#160; kit&#160;</td>
			<td style="width:127px;">Qiagen</td>
			<td align="right" style="width:119px;">74104</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Pellet pestile motor (Tissue homoginizer)</td>
			<td style="width:127px;">Sigma</td>
			<td style="width:119px;">Z359971-1EA</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:293px;">96 well microtiter plates V bottom</td>
			<td style="width:127px;">Thermo</td>
			<td align="right" style="width:119px;">2605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">1X PBS</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:119px;">10010-023</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:293px;">OneComp eBeads</td>
			<td style="width:127px;">eBioscience</td>
			<td style="width:119px;">01-1111-42</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">CD45.2-APC</td>
			<td style="width:127px;">eBioscience</td>
			<td style="width:119px;">17-0454-81</td>
			<td>Working dilution 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Ly-6G-eFlor 450</td>
			<td style="width:127px;">eBioscience</td>
			<td style="width:119px;">48-5931-82</td>
			<td>Working dilution 1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">BSA</td>
			<td style="width:127px;">HyClone</td>
			<td style="width:119px;">SH30574.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">RBC Lysis Buffer (10X)</td>
			<td style="width:127px;">Biolegend</td>
			<td style="width:119px;">420301</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Live/Dead fixable aqua dead cell stain kit</td>
			<td style="width:127px;">Invitrogen</td>
			<td style="width:119px;">L-34957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">EDTA (0.5M)</td>
			<td style="width:127px;">lifetechnologies</td>
			<td style="width:119px;">15575-020</td>
			<td></td>
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			<td height="21" style="height:21px;width:293px;">CD16/CD32 FcBlock</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">553142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Facs tubes polystyrene round bottom tube</td>
			<td style="width:127px;">BD</td>
			<td style="width:119px;">352052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:293px;">Formaldehyde</td>
			<td style="width:127px;">Polyscience</td>
			<td style="width:119px;">4018</td>
			<td></td>
		</tr>
	</tbody>
</table>

intra-tracheal administration of haemophilus influenzae in mouse models to study airway inflammation

여기, 우리는 효율적으로 자세한 절차를 설명하고 직접 헤모필루스 인플루엔자를 제공 마우스의 하부 호흡기로. 우리는 H. 제조 과정을 보여 인플루엔자 접종, H.의 내부 기관 점안 폐에 인플루엔자, 차동 유전자 발현 분석을위한 기관지 폐포 세척액 (BALF), 폐포 세척액에서 면역 세포의 분석 및 RNA 분리의 컬렉션입니다. 이 절차는 임의의 박테리아, 바이러스 또는 진균에 폐 염증 반응을 연구하는데 사용될 수있다. 적은 모호성으로보다 효율적으로 하부 호흡기로 세균 접종을 제공하기 때문에 직접 기관 점안은 주로 비강 내 또는 에어로졸 흡입 절차를 통해 바람직하다.

내 기관 점안은 식염수 설치보다 폐포 세척액 (그림 1A, 왼쪽 패널)에서 백혈구의 현저하게 증가 된 수의 결과. 백혈구의 미분 계수 분석 분명히 증가 호중구 침윤 (도 1, 오른쪽 패널)을 보였다. BALF에서 세포의 FACS 분석은 상기 호중구 수의 증가 (도 1b)를 확인했다. 폐 조직을 H &amp; E 염색 섹션의 조직 학적 분석은기도 염증 (도 1C)을 증가 하였다. 이러한 데이터 집합 내 기관 점안은 생쥐의기도 염증을 유도하는 것이 좋습니다. 기도 기전을 조절하는 신호 전달 경로를 연구를 위해이 방법의 사용을 지원하기 위해, 우리는 E3 유비퀴틴 리가 가려움에 대한 결핍 마우스를 사용 하였다. 가려움은 15 염증성 신호 전달 경로를 조절에 관여하는 것으로 알려져있다. 우리는 접종나이와 성별을 일치 야생형 C57BL / 6 제어 및 가려움는 - / - NTHi와 마우스. - / - 우리는 제어 WT과 가려움의 폐 조직에서 RNA를 격리 마우스와 차별적으로 발현되는 염증 유전자를 식별하기 위해 전체 전 사체 서열 분석을 수행 하였다. 도 2에 나타낸 바와 같이, 가려움 결핍 여러 유전자의 차등 발현 결과. 이것은 인트라 기관 점적은 폐 염증, 유전자 발현 프로필 및 신호 전달 경로를 조사하기 위해 사용될 수 있음을 시사한다. <img alt="그림 1" src="/files/ftp_upload/53964/53964fig1.jpg" /> 그림 1 : NTHi의 인트라 기관 접종 쥐에서 폐 염증을 유도 마우스가 NTHi (106 CFU / 마우스) 또는 식염수 제어 접종 하였다.. 접종 24 시간 후, 마우스 (A) BALF 수집 한 희생시켰다. 총 백혈구, 단핵구 및 호중구 수를 측정 하였다. 데이터 제시으로는 ± SEM을 의미한다. N = / 그룹 4 쥐. * 식염수 처리 된 마우스에 비해 P &lt;0.05를 나타냅니다. (B)의 BALF 세포는 안티 GR1 항체로 염색하고, FACS로 분석 하였다. (C) H &amp; 백혈구 침윤과 염증을 보여주는 폐 섹션을 E-스테인드. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53964/53964fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/53964/53964fig2.jpg" /> 도 2 : WT 가려움의 폐 차등 유전자 발현 - / - 인트라 기관 NTHi 접종 NTHi 접종 후 24 시간이 RNA 두 WT의 폐에서 분리 된 다음 마우스 (1, 2) 및 두 개의 가려움 - / -. (1, 2) 마우스. (A) RNA의 품질 bioanalyzer를 사용하여 분석 하였다. (B) 열 맵에서 멀리 SD의 척도로 해석 (Z-점수를 표시백만 분의 로그 2 수의 평균)을 (가려움에 에저를 사용하여 식별 172 차등 발현 유전자의 2 CPM)을 로그 - / -. WT 마우스에 비해 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53964/53964fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation at JoVE.com

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

의 내부 기관 관리<em&gt; 헤모필루스 인플루엔자</em&gt; 마우스 모델에서기도 염증을 연구하는

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

intra-tracheal-administration-haemophilus-influenzae-mouse-models-to

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

10.1K 조회수.  Baylor Research Institute. We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models. 

비디오: Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

공동 문화 모델 녹농균 Biofilms는 라이브 인간 에어웨이 세포에 들어라

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

연구

면역학 및 감염병학

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

알레르기기도 염증 동안 폐 돌기 세포 성숙과 마이 그 레이션 분석

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

양극화 인간기도 액체 덮인 문화를 구축 호흡기 병원균에 대한 숙주 세포 응답을 공부하는 상피 Calu-3 전지<em&gt; 체외에서</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

생체 내 이미징 방법에 급성 및 만성 염증을 구별하는

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

키메라 마우스 모델에서 재조합 인플루엔자 백신의 비강 관리는 점막 면역을 연구하기 위해

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

인플루엔자 A 바이러스의 핵 단백질과 마우스 MX1 단백질의 공동 면역 침전

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells.
Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5+) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5+ fraction are subsequently stained with &#945;GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 108 iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Here we present an adapted protocol that can be used to generate a large number of murine invariant natural killer T cells from mouse spleen. The protocol outlines an approach by which splenic iNKT cells can be enriched for, isolated and expanded in vitro using a limited number of animals and reagents.

마우스 비장에서 불변 자연 살해 T 세포를 분리하고 확장을위한 최적화 방법

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses). 
The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

복구 시간 증가 및 제한 염증 반응에서 미주 신경 자극 메커니즘을 연구에 LPS 관리를 감소

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,&#160;chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

We describe a method for generating Precision-cut Lung Slices (PCLS) and immunostaining them to visualize the localization of various immune cell types in the lung. Our protocol can be extended to visualize the location and function of many different cell types under a variety of conditions.

정밀 절단 라이브 폐 수지상 세포를 시각화하기 위해 마우스 폐 슬라이스

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal host-microbe interactions. It is well established that different commensals exhibit a different potential to stimulate the host intestinal immune system. Such investigations involve vertebrate animals, especially rodents. Since increasing ethical concerns are linked with experiments involving vertebrates, there is a high demand for invertebrate replacements models. 
Here, we provide a Galleria mellonella oral administration model using commensal non-pathogenic bacteria and the possible assessment of the immunogenic potential of commensals on the G. mellonella immune system. We demonstrate that G. mellonella is a useful alternative invertebrate replacement model that allows the analysis of commensals with different immunogenic potential such as Bacteroides vulgatus and Escherichia coli. Interestingly, the bacteria exhibited no killing effect on the larvae, which is similar to mammals. The immune responses of G. mellonella were comparable with vertebrate innate immune responses and involve recognition of the bacteria and production of antimicrobial molecules. We propose that G. mellonella was able to restore previous microbiota balance, which is well known from healthy mammalian individuals. Although providing comparable innate immune responses in both G. mellonella and vertebrates, G. mellonella does not harbor an adaptive immune system. Since the investigated components of the innate immune system are evolutionary conserved, the model allows a prescreening and first analysis of bacterial immunogenic properties.

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

Here, we provide a detailed protocol for an oral administration model using Galleria mellonella larvae and how to characterize induced innate immune responses. Using this protocol, researchers without practical experience will be able to use the G. mellonella force-feeding method.

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